A recombinant Coxsackievirus B3 (CVB3), v205T, was constructed by inserting repeats of a target sequence for miR-205 between the coding sequence of P1 and P2. Its replication was analyzed in Hela and A549 cells, in which the level of miRNA-205 was known to be low and high, respectively. The results showed that the insertion of miRNA-205 targets in the selected site did not affect the infectivity of the recombinant virus in Hela cells, but greatly reduced its replication in A549 cells, resulting in more than 100-fold reduction in virus titer. miR-205 mimics and inhibitors were used to study the mechanism of differential replication of v205T. miR-205 mimics inhibited the replication and cytotoxicity of v205T in Hela cells, whereas miRNA-205 inhibitors increased the replication and cytotoxicity of v205T in A549. These results showed that the replication of v205T was regulated by cellular miR-205. The work provided basis for using CVB3 vectors for cancer viral therapy and vaccine development.