The message - 'Don't throw it, stow it, take your litter home' - is appearing on six electronic message boards on major roads around the city normally used for traffic information.
It also follows the clean up on two dual-carriageway 'gateway' roads into Gloucester. The council spent thousands of pounds conning off lanes of traffic to remove lorry loads of rubbish dropped by thoughtless motorists and their passengers.
And it coincides with a current anti-litter poster campaign reminding residents that they pick up the£1m bill it costs to keep the city clean.
Andrew Lewis, cabinet member for streetcare, said:' Keeping the city clean and looking after the environment in which we live is one of our top priorities.
'It is important to our residents and we are increasing our armoury in tackling this anti social issue. We need to get the message across that it is a duty on everyone to use litter bins or take their rubbish home.
'When you consider some of the items we find thrown from moving vehicles it is also a major road safety issue as well.'
The messages will be timed to catch commuters, the 'school run', night clubbers and Sunday shoppers.
More information about the council's anti litter campaign can be found at www.gloucester.gov.uk/1million.