The actors co-starred in the long-running sci-fi series, with Takei playing Hikaru Sulu and Shatner as Captain Kirk.
But they have feuded since their days on the Starship Enterprise and last month Shatner told The Times that Takei and other Star Trek actors who had criticised him were "bitter".
Speaking while promoting his book, Boldly Go, Reflections On A Life Of Awe And Wonder, he said: "George has never stopped blackening my name. These people are bitter and embittered."
Takei has now bitten back during an appearance on The Graham Norton Show, where he was talking about the West End musical George Takei’s Allegiance, which is based on his childhood experience of internment in World War II.
Asked if it was true that he and Shatner do not get on, the 85-year-old said: "You are the last chat show host to be allowed to ask that question as it has become so tiresome to talk about.
"When Bill has a book to sell he needs publicity and accuses us of using him.
"My subject is more substantial and important.
"He is a cantankerous old man and I will not talk about him anymore. I vow that this is the very last time I talk about him."
The Graham Norton Show is on BBC One tonight (Friday 2 December) at 10.40pm and is also available on BBC iPlayer.
Watch: 'My heart is heavy': Star Trek actors pay tribute to the late Nichelle Nichols