The Harry Potter author arrived at the Edinburgh event with Better Together head Alistair Darling but declined to comment on her presence.
She took a seat next to him at the No campaign fundraiser in the city's Festival Theatre.
Izzard was greeted with positive heckles as he launched the event with help from Rowling.
A call of "Eddie I love you" greeted the comedian as he took to the stage for his show Scotland, Please Don't Go.
Izzard responded: "A positive heckle, I wasn't expecting that, I was ready for a fight."
However, Alistair Darling did not receive such a positive response, with one audience member shouting "try harder" as he was introduced to the crowd.
Izzard told the audience: "I know some people might be against what I believe but whatever happens to Scotland I hope you have a good time.
"There must be people in Scotland who don't give a damn but I bloody well do.
"I apologise for all the bad stuff, the war, the blood, the death but I'm the only Edward to run eight marathons carrying a Scotland flag.
"It all works and in 300 years Scotland's not lost its identity. I mean if you watch a Scotland football match you can definitely see that you've not lost your identity."
The comedian also asked the crowd to give a round of applause to the nationalist independent MSP Margo MacDonald who died earlier today, adding: "I hope she liked that. I didn't know her but her reputation was huge."
Izzard had admitted that he did feel intimidated ahead of the show, which is raising funds for Better Together.