Abstract: The effect of alternated propagation of nucleopolyhedroviruses(NPV) in two insect hosts on virulence was firstly investigated. The Spodoptera litura NPV(SlNPV)and Autographa california NPV (AcNPV) were mixed equally to infect S. exigua and S. litura, respectively, and after that, they were propagated in the original host successively or in S. exigua and S. litura alternately. And the virulence of NPVs of each generation were compared. The results showed that alternated propagation in two different hosts significantly improved the virulence of NPV. NPV propagated in S. litura were more effective against S. exigua after one passage in S. exigua, and its efficacy on S. litura did not decrease. And it was the same to NPV propagated in S. exigua againt S. litura. NPV propagated in S. exigua was also more effective against S. litura after one passage in S. litura, and its efficacy on S. exigua did not decrease. All the results indicated that, compared to the efficacy of NPV after serial passages in same host, the efficacy of NPV against non-host target was improved after alternated propagation of mixed NPVs in two different hosts.