Powered by biomethane gas, the Bio-Bus will use waste from more than 32,000 households along its 15-mile route.
Operated by bus company First West of England, the bus will fill up at a site in Avonmouth, Bristol, where sewage and inedible food waste is turned into biomethane gas.
The bus, which can seat up to 40 people, was unveiled in the Bristol area last autumn.
Transport company First is showing off the bus in Bristol on Tuesday and it will operate four days a week on Service 2, which stretches from Cribbs Causeway to Stockwood, from 25 March.
If the route proves a success, First will consider introducing more "poo buses".
First West of England managing director James Freeman said: "Since its original unveiling last year the Bio-Bus has generated worldwide attention and so it's our great privilege to bring it to the city, to operate - quite rightly - on Service 2.
"The Bio-Bus previously made an appearance running between Bath and Bristol Airport at the end of last year, but it's only actually been used once before in the centre of Bristol itself."
He went on: "The very fact that it's running in the city should help to open up a serious debate about how buses are best fuelled, and what is good for the environment."
From Sky News