The unprecedented closure of the second-biggest US subway network will have an impact on hundreds of thousands of commuters.
Officials will carry out an inspection of power cables after a fire caused major delays earlier this week.
Last year, a similar incident caused a train to fill with smoke in a tunnel, killing one passenger.
The 119-mile (230-km) network, known as Metro, will be closed from midnight on Tuesday (0400 GMT) to 0500 local time on Thursday.
All six lines and 91 stations will be affected. Some 700,000 people use the network daily.
Safety teams will inspect about 600 underground electrical cables, Metro general manager Paul Wiedefeld said in a statement.
"While the risk to the public is very low, I cannot rule out a potential life safety issue, and this is why we must take this action immediately," Mr Wiedefeld said, without ruling out further disruptions.
Officials said the closure would be the first shutdown of the service that is not weather related since operations began in the 1970s.
Despite the interruption, federal government offices are expected to open as usual.
Meanwhile, taxi-hailing app Uber said it would cap surge pricing in the Washington area at 3.9 times base fares.
Ride-sharing service Lyft announced it would offer new users $20 (Â£14) off their first ride during the closure.
Washington DC's subway system will be shut down for 29 hours from Tuesday night for emergency safety checks.