The selectively oncolytic effects of mtHSV, a HSV icp34.5 mutant with lacz gene insertion, on several tumor cells in vitro and its antitumor effects by the intratumoral (IT) route to nude mice loaded the human hepatoma xenografts were explored. The mtHSV could conditionally replicate in and lyse Hep-3B (human hepatoma cells), Hep-2 (human larynx cancer cells) and SPC-A1 (human lung cancer cells), but not MRC-5 (human fibroblast cells). The 125 nude mice loaded with Hep-3B were randomly divided into five treatment groups and given three IT injections with three different dose of the mtHSV, adriamycin (ADM), or vehicle (supernatant of non-infected Vero cells). Significant tumor growth inhibition (30%-70%) was seen in the nude mice treated IT with mtHSV, whereas tumors treated IT with Vero supernatant displayed rapid tumor growth. The results of regular and biochemical blood examination, systemic necropsy and pathological slices showed that mtHSV almost has no side effect on treated mice. RT-PCR results revealed that the replication of mtHSV was exclusively confined to the treated tumors, but not to other organs. Our results provide further preclinical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.