Objective To construct replication-defective recombinant adenovirus type 5 (rAd5) vector containing Zika virus (ZIKV) envelope protein prM-E, and determine the expression of prM-E in cells and its immunogenicity in mice. Methods prM-E gene fragment was obtained from ZIKV strain Z16006 (Asian type),recombinant adenovirus rAd5/prM-E was enveloped with recombinant adenovirus AdMax^TM system. C57BL/6 mice were immunized by intramuscular injection of rAd5/prM-E at three doses(10⁷, 10⁸ and 10⁹ PFU), mice were immunized again at the same dose at week 3, blood was taken from the eyeballs of mice and spleen lymphocytes were separated at week 5. Humoral and cellular immune response of mice to ZIKV prM-E were detected by ELISpot and ELISA respectively. Results The replication-defective recombinant adenovirus vector rAd5/prM-E was constructed successfully, expression products of 293A cells infected with rAd5/prM-E were detected by Western blot and anti-ZIKV E antibody, 56 kDa protein band which corresponded with E protein was observed. Spots forming cells (SFCs) secreted by mice splenic lymphocyte specific IFN-γ was detected by ELISpot method, results were (688.54±186.43), (1 084.90±144.14), and (1 640.20±147.13) SFCs/10⁶ splenic cell respectively, which were positively proportional to the immune dose; anti-E antibody in serum of immunized mice was determined by enzyme-linked immunosorbent assay (ELISA), and the titers (log10 value) were (3.14±0.39), (3.50±0.30), and (3.74±0.25) respectively, all were significantly higher than(0.80±0.17) of control group (P<0.001). Conclusion rAd5/prM-E has the ability to infect mice and induce strong specific antibodies and cellular immune response, which indicates that prM-E has good immunogenicity and provides a reliable immune source for the development of ZIKV candidate vaccine.