The M74 will be closed in both directions between J4 Maryville and J5 Raith from 20:00 on Friday 18 November to 06:00 on Monday 21 November.
It is for the safe demolition of Bothwellpark Road Bridge near Hamilton.
The work is part of an ongoing Â£500m works programme to improve Scotland's motorway network.
Graeme Reid, Transport Scotland project manager for the M8, M73, M74 Motorway Improvements Project, said: "This is the most significant road closure we have implemented as part of the works and we are anticipating major delays on all approaches, the surrounding roads and the main diversion route.
"We are advising road users to plan their journey, avoid the area and surrounding local roads and, if possible, to use alternative modes of transport for the weekend."
The weekend closure will result in no access from the M73 to the M74 southbound at J4 Maryville or to the M74 northbound at J5 Raith.
Signed diversion routes will be in place via the A725, A8 and the M73, although Transport Scotland is warning road users to expect major delays on all surrounding routes.
Mr Reid added: "On a typical weekend we can expect to see as many as 140,000 vehicles using this main arterial route, and we fully appreciate the disruption this will cause.
"However, we need road users to play their part by avoiding the area where possible and using alternative modes of transport to keep traffic levels on the diversion route to an absolute minimum."
Motorists will continue to have access to the M74 southbound at J5 Raith and the M74 northbound at J4 Maryville and J3A Daldowie.
However, there will be no access to Bothwell Services from the M74 southbound.
The next available service area is the M74 southbound at J11 Happendon and J13 Abington.
In a bid to reduce congestion on alternative routes, South Lanarkshire Council will put in place parking restrictions on a short section of Main Street, Bothwell, for the duration of the weekend closure.
ScotRail will also provide extra capacity on trains on Lanarkshire railway services throughout the weekend.
A busy section of Scotland's motorway link to England is to close for a full weekend next month causing "significant delays" to drivers.