The 43-year-old MP appeared after summons were issued by the department, as a Delhi court probing the 2G spectrum allocation exempted her from personal experience.
IT officials said on condition of anonymity that today's enquiry focused on the Rs 200 crore transaction from DB Realty, whose proprietor Balwa is a co-accused in the 2G spectrum case, to Kalaignar TV.
They said they may question her again if required.
Kalaignar TV Managing Director Sharad Kumar was also questioned, officials said. Both were questioned by a Deputy Director level officer, sources added.
The DMK leader, at the end of the one-and-a-half hour questioning, left the IT office through a rear exit.
A Delhi court had allowed Kanimozhi and Kumar exemption from personal appearance even as it is slated to pronounce its verdict on their bail plea on May 14, just a day after the counting of votes for 234 Assembly seats in Tamil Nadu, which went to polls on April 13.
Both Kanimozhi and Kumar had already appeared before the Enforcement Directorate office in New Delhi in connection the 2G spectrum case.
The ED had summoned them for questioning about the transfer of Rs 200 crore to Kalaignar TV, in which Kanimozhi and Kumar hold a 20 per cent stake each.
Karunanidhi's wife Dayaluammal, who has been named a witness in the CBI supplementary charge sheet, holds 60 per cent stake in Kalaignar TV.