The strike means no service on the Piccadilly line — and no Night Tube until Saturday 29 September when another strike is due to begin.
The RMT has apologised to passengers for the disruption and say the strikes "revolve around our working conditions and management breaking our agreements."
"We are striking to be treated with dignity and respect at work."
The chaos caused by the strike also reached the UK's biggest airport, London Heathrow which is served by the Piccadilly Line.
Meanwhile, the person working for the Piccadilly Line Twitter account admitted they did not know the reason for the strike while other social media users have hit out at London Mayor Sadiq Khan who promised there would be no strikes while he was mayor.
Even Piccadilly line staff don't know why they're on strike. You couldn't make this up #RMT#tubestrike 😂😂 https://t.co/SrN8WwvuS3