A dedicated southbound cycling lane will be created for around 300 metres outside Federation Square and over Princes Bridge.
Chair of the Transport portfolio, Councillor Nicolas Frances Gilley, said the bike lane will increase pedestrian and cyclist safety ahead of further works on the CBD security upgrade project.
"The two-metre wide bike lane we're creating will replicate the traffic conditions already seen on the northbound side of Princes Bridge," Cr Frances Gilley said.
"With the Metro Tunnel works already closing one of the two left turn lanes from Flinders onto St Kilda Road, the impact of these changes on vehicular traffic will be negligible."
The number of traffic lanes on the southbound side of Princes Bridge will be reduced from two lanes to one, with tram services along Swanston Street not impacted by the works.
Line marking works to create the new lane will be coordinated with the Metro Tunnel Project works to create an entrance to Town Hall Station at Federation Square.
Cyclists can continue to use the existing bike lane on the Princes Bridge footpath while the new bike lane is installed over three nights from Monday, 22 October 2018.
"The new lane will take cyclists off the Princes Bridge footpath, increasing safety for pedestrians," Cr Frances Gilley said.
"The protected pedestrian zone on Princes Bridge will be expanded, with the existing water-filled barriers moved to the kerb line, ready for them to be upgraded to permanent barriers in early 2019."
Further work will be conducted by the City of Melbourne and the Victorian Government to install protective bollards and barriers around an expanded forecourt at Flinders Street Station over the coming months.
The City of Melbourne will work with the Victorian Government to minimise disruption as much as possible during the works.