Nikolai Peskov told the pranksters that "it is not quite right" for him to be enlisted.
The son of Kremlin spokesman Dmitry Peskov refused to join Russia's army in a prank call with opposition activists.
The viral stunt was released online shortly after Russian President Vladimir Putin announced a "partial mobilisation".
Dmitry Peskov told reporters that he was "aware of it" but stated that the full transcript of the phone call had not been published.
The hoax was conducted live by Dmitri Nizovtsev, host of "People's Politics" YouTube channel that was created by supporters of jailed Russian opposition figure Alexei Navalny.
In the viral video, Nizovtsev called Nikolai Peskov, posing as an army recruiter in charge of calling up reservists to fight in Ukraine.
"A summons [for medical examination] was sent to you today," Nizovtsev said in a firm voice to Nikolai Peskov.
But the 32-year-old implied that he would refuse to be enlisted, stating "of course, I won't come tomorrow at 10:00."
"You must understand, if you know that I am Mr Peskov, how wrong it is for me to be there," he added, apparently disconcerted.
"I will deal with it on another level," Nikolai said, before stating that he is not a volunteer "rank and file".
"I have no problem defending my homeland - but I need to understand the expediency of my appearance there, I am talking about certain political nuances."
Moscow has said that the "partial mobilisation" would only involve300,000 reservists who have "military experience" but some citizens are fleeing the country following Putin's speech.