The Kingsway Tunnel in Wallasey will be closed in both directions overnight from 7pm on Friday.
It will re-open at 6.30am on Saturday.
The Queensway (Birkenhead) Tunnel will remain open as usual.
The night time works involve removing a major overhead gantry sign which spans the Wallasey plaza end of the Kingsway Tunnel.
Neil Scales, Chief Executive and Director General of Merseytravel, which owns and operates the two Mersey Tunnels said: “We looked at every possible option to maintain access through the Kingsway Tunnel but the only safe way to remove the gantry is to put this full closure in place.
“We have done what we can to limit any potential impact on traffic by timing these works overnight.
"The 7pm start for the works is the latest we can start them if we are to ensure the works are carried out safely and we can inspect the road surface so the tunnel can be reopened at 6.30am on Saturday morning.
“We’ve done what we can to notify people to make alternative travel arrangements, however, we apologise if this does cause people some inconvenience.”
As a result of the closure, all class 1 and class 2 traffic (cars; light vans; vehicles with two or less axles; and vehicles seating nine or less people) will be diverted to the Queensway Tunnel.
Unfortunately, Heavy Goods Vehicles (class three and class four traffic) are not permitted to use the Queensway Tunnel and, therefore, will be diverted via the Runcorn Bridge.