Williams' 1984 hit "All My Rowdy Friends Are Coming Over Tonight" was altered and had been used to open the Monday Night Football telecast on ABC and ESPN since 1991.
However, ESPN pulled the song from this past Monday's game between Tampa Bay and Indianapolis after Williams made an analogy with Adolf Hitler and President Barack Obama earlier that day on a Fox News show.
On Thursday, the network decided to end the relationship altogether.
"We have decided to part ways with Hank Williams Jr.," ESPN said in a statement. "We appreciate his contributions over the past years. The success of Monday Night Football has always been about the games and that will continue."
Williams, on the Fox News program "Fox & Friends," said of President Obama playing golf with House Speaker John Boehner: "It'd be like Hitler playing golf with (Israeli Prime Minister Benjamin) Netanyahu."
The 62-year-old Williams also said Obama and Vice President Joe Biden were "the enemy."
On Tuesday, Williams tried to explain his comments.
"Some of us have strong opinions and are often misunderstood," Williams wrote on his personal website. "My analogy was extreme -- but it was to make a point. I was simply trying to explain how stupid it seemed to me -- how ludicrous that pairing was. They're polar opposites and it made no sense. They don't see eye-to-eye and never will. I have always respected the office of the President."
Williams, on Thursday, said the decision to pull the song permanently from Monday Night Football was his and not the network's.
"After reading hundreds of e-mails, I have made MY decision," he wrote again on his website. "By pulling my opening Oct 3rd, You (ESPN) stepped on the Toes of The First Amendment Freedom of Speech, so therefore Me, My Song, and All My Rowdy Friends are OUT OF HERE. It's been a great run."
Williams won Emmy Awards from 1991-94 for his Monday Night Football theme.