The 28-year-old Du Toit won three gold medals at the Aquatics Centre during the London 2012 Games, taking her tally to 13 across three Paralympics.
She was denied the golden send off she had hoped for last night ahead of her retirement, as she finished her career with silver in the women's 100m Freestyle - S9 final, behind Australia's Ellie Cole.
Afterwards, the emotional South African fought back the tears as she reflected on her achievements in a remarkable two decades in the pool, but she insisted she had no regrets.
'It's not the medal that's emotional,' she said. 'It's knowing that I'll never walk out again [to the pool]. Seeing the youngsters doing so well and coming up, that's really special.
I walk away knowing I have no regrets. Hopefully I can show to the rest of the world that it is possible to make a success twice in a row.Natalie du Toit
'It's sad, I've been there for 22 years. I walk away with a couple of [London 2012] medals - I walk away knowing I have no regrets.
'I know that I haven't come in prepared [for the Games] - to win those medals is special for me. To walk away from the last race with a medal was something that I wanted to achieve.'
As she plans her next move, Du Toit is still undecided as to what the future holds, but she is ready to take the same determination she showed throughout her Swimming career into whatever challenge awaits her.
'I'm a bit nervous because I don't really have anything else to do other than a bit of sponsor work,' she added. 'I don't know what I'm good at, or what I want to do one day, but I promised myself it's time to move on.
'Hopefully I can show to the rest of the world that it is possible to make a success twice in a row.'