The Gandhi scion appeared before civil judge A I Sheikh, who read out the charges levelled against him and also the statement of the complainant Rajesh Kunte, an RSS activist.
The defamation case was filed Kunte following Gandhi's utterances at a rally in Thane in March 2014 in which he had accused the RSS of killing Mahatma Gandhi, the Father of the Nation.
After reading out the complaint against Gandhi, the judge asked him, "Do you accept the charges?"
To this, Gandhi replied, "I plead not guilty."
The court then proceeded to frame charges against the Congress leader under IPC sections 499 (defamation) and 500 (punishment for defamation).
Under section 500, a person found guilty of defamation faces simple imprisonment for a term which may extend to two years, or with fine, or with both.
The court fixed August 10 as the next date of hearing, when the actual trial will begin.
Emerging from the court, Gandhi said if need be, he would be present in the court again.
"Let them (the BJP and the RSS) slap as many cases as they want against me. Ours is a fight of ideology. We will fight them and win," he told reporters outside the court.
"Ours is a fight of ideology and system. Let them file any number of court cases against me, I shall fight them, I am not bothered." he said.
(With inputs from agencies)