Chicken infectious bronchitis virus (IBV) causes severe respiratory and renal dysfunction in chickens. In the present study, we used reverse transcription-polymerase chain reaction (RT-PCR) to amplify the nucleocapsid (N) protein gene of strain Massachusetts (Mass) H120 of IBV that is commonly used in the vaccine production in Iran. The PCR product with the expected size of 1.2 kb was cloned into a TA-vector and subsequently sub-cloned into expression vector pET-23a(+). The recombinant plasmids were transformed into competent BL21(DE3) strain of E. Coli. The recombinant N protein was expressed as a soluble fusion protein tagged at its C-terminal with 6 x His residues. In Western blot analysis, a convalescent serum from a chicken tested positive by a commercial IBV detection kit specifically reacted with the recombinant protein. The IBV negative chicken serum did not react with the recombinant protein. In a reciprocal experiment, antiserum raised against the purified fusion protein reacted with the corresponding proteins of strains H120 and 4/91. This is the first report of cloning and expression of the N protein gene cDNA of strain Mass H120 of IBV in a bacterial expression system. The potential uses of the cloned gene and the recombinant N protein have been discussed.