Canine distemper virus H gene is immunogenic and induces neutralizing antibodies against Canine distemper virus (CDV). In the present study the recombinant plasmid pSin.cdv.h transfected HeLa cell expressed H protein which was confirmed by immunoperoxidase test. The in vivo immunological response was studied in dogs immunized through IM route. The immune response on 30th day post vaccination induced by 4μg dose of rplasmid revealed protective serum neutralizing antibody titer 120, while control group did not show any antibody titer against CDV. Stimulation Index (SI) was found higher in vaccinated group as compared to healthy control animals. The T-cell response in the blood of vaccinated dogs suggested the role of cell mediated immunity in protection apart from neutralizing antibodies in serum. The challenge test revealed 100% protection of vaccinated dogs while all control dogs died of cdv infection. Therefore, this study demonstrates that DNA vaccination with 4μg dose of pSin.cdv.h can provide protective immunity 30 days post vaccination.