The warrant was issued by Mettur judicial magistrate-II S. Sreedharan.
Several cases have been filed in lower courts in the state against Khushboo following her media interview about the rising trend of pre-marital sex in the state.
Her remarks were widely criticised by many political parties, which alleged that the actress had hurt Tamil sentiments.
The complaint against the actress was filed in the Mettur court by a Pattali Makkal Katchi functionary, Murugesan.
Khushboo's lawyers had sought exemption on her personal appearance in the defamation case at the Mettur court because of "prior social commitments", which the court rejected Friday.
The court said, her plea was prepared before hand and in it she had said she would be in Mumbai.
Hearing in the case has been postponed to Dec 16.
The Madras High Court had exempted Khushboo from appearance in two other lower courts earlier this week.
The Madurai Bench of the Madras High Court Oct 25 had stayed the implementation of a warrant issued by a Tiruchirappalli court for her appearance in another defamation case for the same remarks.
Both the Madras High Court and its Madurai Bench had also allowed petitions filed by her for exemption from personal appearances in courts.
The Madras High Court Wednesday stayed proceedings against Khushboo for alleged violation of social norms in magistrate courts in Tiruvannamalai and Ariyalur districts of Tamil Nadu, following her statements on pre-marital sex in a leading magazine in September.
The actress had said in the interview that it was fine for girls to indulge in pre-marital sex as long as they took precautions. She later justified her statement by saying that no educated man expected his partner to be a virgin.
On Wednesday, Khushboo told the high court that "persons inimical to her name and fame" had filed the complaints though she had only expressed her personal opinion in the context of gender equality and the status of women in society.
Khushboo had reiterated Wednesday that she did not have "any intention to insult or cause injuries to the feelings of any section of any specific community or to defame the traditions, culture and customs of the state".
However, Suhasini Maniratnam, wife of the noted director Maniratnam, had spoken up in support of Khushboo at the opening of the International Film Festival of Tamil Nadu Wednesday.
After this, PMK leader Kasambu Poomalai has reportedly said: "Suhasini must apologise at once. Otherwise, we will file criminal cases against her as well."