The Clark Memorial Bridge closes at midnight on Tuesday for construction of the downtown crossing of the Ohio River Bridges Project.
Project spokeswoman Mindy Peterson says the bridge that connects Jeffersonville and downtown Louisville will close for about six weeks.
Nearly 25,000 vehicles cross the bridge every day.
Peterson says there will be no changes to the Clark Memorial itself but closing the bridge is necessary to work on the downtown crossing.
An exact date for reopening the Clark Memorial Bridge has not yet been set, but officials estimate it will be sometime in mid-August.
Motorists are encouraged to use the Kennedy Bridge while the Clark Memorial is closed, and pedestrians and cyclists should use the Big Four Bridge.
TARC officials say buses that cross the Clark Memorial will be re-routed to the Kennedy.