In an email to the league, Chairman Dave Massam cited a lack of availability among players as the reason for their resignation, adding they didn't want to start a season they may not be able to complete.
Chairman Andy Hosker said: "We have unfortunately today received notice from Dave Massam at Southport Hesketh tendering their resignation from the WLL.
"This is clearly extremely disappointing news to receive, Southport Hesketh have been members of the league for many years (previously as Fleetwood Hesketh) and I’m sure everyone will be disappointed that they will no longer be with us.
"Any planned fixtures issued will now be postponed.
"This means that Division 1 will only have 15 teams during the season 2025/26."
Manager Denis Hill was awarded the Wilf Carr Loyalty Trophy at the league's Presentation Night in June.
The club were runners-up of Division One in 2008/09 and the Richardson Cup in 2014/15.
Striker Anthony Wong twice won the league Golden Boot award for top scorer in 2008/09 and 2010/11.
Meanwhile, Caton United Reserves will no longer compete in Reserve Division Two.
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