To study the effect of miR-490 on Coxsackievirus B3 (CVB3) replication, HeLa cells were trans- fected with miR-490 in vitro, and infected with a Renilla luciferase (RLuc)-expressing CVB3 variant (RLuc-CVB3). The activities of RLuc in these cells were measured at 8h intervals from 0 to 40 h post-infection (p.i.), and the effects of miR-490 on RLuc-CVB3 replication were observed. In a further study, HeLa cells were transfected with either miR-490 or antisense miR-490 (AMO-miR-490), and were then infected with an enhanced green fluorescence protein (EGFP)-expressing CVB3 variant (EGFP-CVB3). The replication of EGFP-CVB3 was then determined by detecting the expression of EGFP. We observed that miR-490 could significantly inhibit the expression of RLuc in infected cells at 32 h p. i. Furthermore, in HeLa cells infected with EGFP-CVB3 at 32 h p.i., EGFP expression was also significantly inhibited by the presence of mniR-490. The inhibitory effect of miR-490 on EGFP expression in EGFP-CVB3-infected cells could be reversed by tranfection with AMQ-miR-490. These results indicated that miR-490 significantly inhibits the replication and expression of QVB3.