The 28-year-old Du Toit won three gold medals at the Aquatics Centre during the Games, taking her tally to 13 across three Paralympics, but she signed off with silver in the women's 100m Freestyle - S9 final as Australia's Ellie Cole won gold in 1:02.77.
World record holder Du Toit, a winner of the event at Athens 2004 and Beijing 2008, finished 0.68 seconds behind in 1:03.45 to take a second silver medal of her Paralympic career.
'It is all over,' said a tearful Du Toit, as she reflected on a 14-year career as an international swimmer.
'I gave everything in the pool, I gave everything as a person. I look back and I'm satisfied. It's time to move on.
'I went out there, tried my best and to get a medal was something special.
'Ellie's a great competitor and to go out there and get second is a little bit disappointing, but it's still a medal.'
Cole claimed her third gold of London 2012 and had been tearful before the race as Du Toit announced her imminent departure.
'It was an amazing swim - everything went to plan,' the 20-year-old said. 'I feel really bad about beating Natalie. I know I should be stoked. I wanted her to do really well.
'I am really going to miss her. She has been such a great support over the last six years and a great mentor and she has taught me so much about Paralympic Swimming.'
Cole's fellow Australian Matthew Cowdrey won his 13th Paralympic Games gold with victory in the corresponding men's event, while Bradley Snyder of the USA won his second gold of London 2012 in the men's 400m Freestyle - S11, a year to the day after being blinded by an improvised explosive device while serving with the US Navy in Afghanistan.
On the track, Irishman Smyth followed in the footsteps of Usain Bolt by completing a London 2012 sprint double.
The 25-year-old, the fastest Paralympian in the world, added the 200m - T13 crown to his 100m title with another dominant display.
And he went a step further than the self-confessed Jamaican 'living legend' Bolt, by smashing the world record for the third time in four races at the Olympic Stadium.
The only one in the final to have gone under 22 seconds, the Irish sprinter - who trains in Florida with USA star Tyson Gay - clocked 21.05. That was 0.90 ahead of Alexey Labzin of Russia and 0.38 quicker than Smyth's old world-best mark.
Another Russian, Artem Loginov, took bronze in a personal best of 22.03.
Meanwhile, Oscar Pistorius showed he is in no mood to let his Paralympic 400m - T44 crown go the same way as his two other individual track titles.
The South African has this week lost his 100m and 200m titles but the 400m is the event in which he reached the Olympic semi-finals and, judging by the two heats tonight, he is looking strong for gold tomorrow.
The 25-year-old ran a strong first half of the race before he was able to ease up long before the line, coming home in 48.31.
Italy's Alex Zanardi claimed his second Paralympic gold medal with victory in the men's H4 Cycling Road Race at Brands Hatch.
The 45-year-old now has two wins at the Kent motor-racing circuit where he first raced as an F3000 driver 21 years ago before losing his legs in an accident in 2001.
Zanardi, who won time-trial gold on Wednesday, won the 64km race in a sprint finish ahead of South Africa's Ernst van Dyk.
China won two of the four Table Tennis titles on offer, in the men's Team - Class 3 with a 3-2 victory over Germany before retaining the women's Team - Class 1-3 title by beating Republic of Korea 3-0.
Slovakia beat France 3-0 to win gold in the men's Team - Class 1-2 while Poland won the other gold on offer with a 3-0 defeat of Spain in the men's Team - Class 6-8 final.
The final of the Mixed Individual - BC3 Boccia will be an all-Republic Korea affair after semi-final wins for Choi Ye-Jin and Jeong Ho-Won while Britain's David Smith will go for gold against Thailand's Pattaya Tadtong in the Mixed Individual - BC1 final.
The Wheelchair Rugby semi-finals will see the USA play Canada and Japan meet Australia after all four recorded victories today.
In 7-a-side Football, Russia beat Brazil 3-1 to reach the final, where they will play Ukraine, who beat Iran 2-1.
The imperious Esther Vergeer won her fourth straight Paralympic Games gold and a 470th straight match as she beat fellow Dutchwoman Aniek van Koot, the world number two 6-0 6-3 in the women's Singles final, while Sweden's Stefan Olsson and Peter Vikstrom won men's Doubles gold.
China won the Wheelchair Fencing women's Team - Open event after seeing off Hungary 45-38 in the final at ExCeL.
Akiko Adachi scored the only goal as Japan claimed women's Goalball gold ahead of China at the Copper Box before Finland were convincing 8-1 winners over Brazil in the men's final.
Germany beat Australia to win the women's Wheelchair Basketball gold.
Mareike Adermann top scored with 19 points for Germany, who led by six after the third quarter before pulling away in the fourth.
The Netherlands beat the United States 71-47 to claim bronze.
China won the women's Sitting Volleyball with a 3-1 victory over the USA in the final while Ukraine beat Netherlands to take bronze.