The NRL Footy Show first aired in 1994 and won 11 Logies for 'Most Popular' show on Australian TV.
The sports program's axing follows poor ratings, a later time slot and budget cuts.
It's unclear what the cancellation means for the hosts future at Channel Nine.
Nine's Director of Sport Tom Malone said: "It's been an incredible ride for The Footy Show. A quarter of a century of laughs, controversy and first-class footy analysis.
"I want to thank everyone who has contributed to this amazing era of live sports-entertainment television – from producers, editors, camera operators, the players, guests and of course the legendary hosts.
"Fatty holds the record for the longest serving host of any prime time Australian TV show – a tribute to both his understanding of the game, his authenticity, and not to mention his incredible comedic timing.
"In recent years, Erin has equally brought a new energy, perspective and showed her passion for the game."
Regular host Beau Ryan also took to Twitter to share his response to the sad news.
He wrote: "Thank you 2 everyone that contributed to the Footy Show over the last 25 years.I grew up idolising Matty Johns, Sterlo, Chief & to work with Fatty was a dream come true.
"Erin & Big marn are legends.Thanks 4 the memories, & if anyone needs me I’ll be halfway up the Centrelink queue.."