The complete closure of the bridge came into force at midnight and will remain in place for at least the next three weeks.
The total closure of the bridge – which is also shut to cyclists and pedestrians – is expected to cause significant disruption to commuters, particularly during the peak rush-hour periods.
Transport Scotland said the decision had been taken as a “precautionary step”.
The Forth Road Bridge operators tweeted that it was “to allow for essential maintenance”.
Confirming the move in a statement, Transport Scotland said: “A full closure of the bridge is being put into effect after midnight on Thursday December 3, 2015.
“The Forth Road Bridge is to shut following the discovery of defective steelwork earlier this week.
“The decision has been taken as a precautionary step after inspections carried out earlier this week showed a fault in the bridge steelwork.
“The results of earlier inspections will continue to be analysed overnight.”
— Traffic Scotland (@trafficscotland) December 4, 2015
Earlier this week, traffic on the bridge was restricted to a single lane in each direction, following the discovery of a defect to a steel component beneath the southbound carriageway. It was found by engineers on Tuesday during a routine planned inspection.
In light of the full closure, bridge users have been urged to avoid travelling if they can.
Those who have to travel have been advised to plan their journeys in advance and to divert via Kincardine or the A9 and M9.
Transport Minister Derek Mackay told BBC Scotland: “In light of the latest information and advice, we’ve been recommended to close the Forth Road Bridge.
“The reason for that is that further traffic will cause further damage to the bridge, having identified the fault, therefore the bridge has to be closed.
“I know that that causes significant disruption and I thank travellers and drivers for their patience but we have to close the bridge for reasons of safety to undertake investigation and repairs, and therefore I would advise the travelling public to avoid travelling if they were going to use the Forth Road Bridge.”
A ScotRail spokeswoman said: “We are working to see what we might be able to do to add more capacity to what is already an extremely busy route.
“However, even if we do manage to add carriages or services in the area, trains are likely to be incredibly busy at peak travel times.
“So if you don’t need to travel, then it’s probably best that you don’t.”
Further updates are expected to be provided by the authorities today.
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