Archant is replacing Clacksons with regional sales house Mediaforce, which will now handle its national ad sales.
Although Clacksons represents other regional publishers such as Tindle Newspapers, Bullivant Media and Iliffe News & Media, the Archant contract is one of the company's larger contracts.
It is not known how the loss of the Archant contract will affect Clacksons moving forward, but sources close to the company insist its future is secure.
Adrian Worsley, managing director of Clacksons, declined to comment on the move.
Clacksons has handled Archant's ad sales since the company was renamed from Eastern Counties Newspapers Group in 2000, but has worked with some of the group's newspapers for as long as 30 years.
The appointment of Mediaforce follows a competitive pitch earlier this year. Mediaforce has been appointed to handle the national ad sales across Archant's portfolio of newspapers, magazine and digital offerings.
The contract includes the national ad sales for four daily regional newspapers, more than 50 weekly newspapers, around 80 magazines, and approximately 200 websites and digital products.
Adrian Jeakings, chief executive, Archant, said: "During the last six months, we have been conducting a thorough review of our arrangements for selling national advertising.
"That process led us to the conclusion that Mediaforce was best positioned and had the scale and resources to progress our engagement with national advertisers in a much more competitive media landscape.
"We are very grateful for the good work that Clacksons has done for us."
Independent ad sales firm Mediaforce currently represents more than 400 publications from 37 regional media groups, including Johnston Press, Northcliffe Media, DC Thomson and parts of Guardian Media Group.
Lawrie Procter, managing director, Mediaforce, said: "Archant really make their products relevant to their audiences. They truly understand their communities, be it rural Suffolk or central London.
"This, coupled with their willingness to work with us to develop new platforms and solutions for advertiser partners, makes this a tremendous opportunity for national brands to engage with."
Procter said Mediaforce is also "really keen to help" Archant realise the value of its "very strong digital assets".
Mediaforce previously handled all the ad sales for Daily Mail & General Trust’s Northcliffe Media but the main newspaper contract moved to Trinity Mirror’s Amra on 1 May. Mediaforce continues to represent Northcliffe’s digital products.