The Tottenham star had believed that Anne-Marie Moore’s child Joshua-James, who was born in February, was his first child.
The pair had had a brief relationship, although they are no longer together.
"Following a paternity test, Jermain Defoe has confirmed that he is not the father of the baby born on February 19 2013," said the player's spokesman.
"Jermain vowed to support the mother after she claimed she was expecting his child but DNA test results have proved that the baby is not his.
"He will be making no further comment."
Defoe had made mention of the child in the run-up to his side’s matches against Internazionale in the Europa League, revealing his joy to be a father for the first time.
"I have a little boy now, so I want to work hard to be a better person and keep on doing what I'm doing," he said ahead of playing Inter Milan in the Europa League.
"I spend a lot of time with him. I see him every day, feeding him, putting him to sleep, changing nappies. I do everything. It's the first but it has been a great experience.
"It's amazing to be honest. Anyone with kids can understand what I'm saying. It changes your mentality, the way you think. I just feel blessed."
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