In order to express and characterize the biological function of protein Reg3A, the partial coding sequence without signal peptide of Reg3A gene were sub-cloned into vector of pET-32a to construct recombinant prokaryotic expression vector pET-32a-Reg3A. After induction with IPTG, the strain of E. coli BL21-Codonplus was transformed successfully with recombinant constructs, which was found to be expressing the recombinant protein in high yield and existed in the form of inclusion bodies. The inclusion body dissolved in urea was refolded into natural conformation after dialysis. The expressed protein was purified by Ni-NTA column. The purified protein, about 95% purity, was confirmed by Western blot. It was further demonstrated that the recombinant protein could effectively inhibited growth of Gram positive bacteria, which indicated that our recombinant Reg3A retained antibacterial activity. The cloning and expression of the Reg3A proteins provide basis for further characterization of the Reg3A biological function.