The selectively oncolytic mtHSV, a HSV icp34.5 mutant with lacz gene insertion, was proved that it was targeted for treating tumors but not other organs, however, its oncolytic mechanism is under confirmation. The results showed that HeLa cells could be lysed efficiently by mtHSV in vitro. In the flow cytometry and Western blot experiment, Ras protein was obviously downregulated on plasma membrane (PM) while the whole Ras protein didn't change along with upregulation of reticulon 3(RTN3) protein at 48 h post infection of mtHSV in HeLa cells. Expression of Ras protein on PM and whole Ras protein in HeLa cells was downregulated by siFTa (inhibitor of a subunits of human farnesyltransferase with siRNA) and siRTN3(inhibitor of RTN3 with siRNA) respectively, and HeLa cells could be killed effectively by siFTa and siRTN3 at 48 h post transfection. So siFTa and siRTN3 effectively suppressed mtHSV infection of HeLa cells. Further, experiments were made to study the relationship between Ras and RTN3 using confocal colocalization and coimmunoprecipitation. The results exhibited that Ras could interact with RTN3 at endoplasmic reticulum. The data put forward that Ras/RTN3 is an important access to HeLa cells for mtHSV. The molecular interaction between Ras and RTN3 may further improve the understanding of the function of Ras and RTN3 in mtHSV infection. The results provide further theoretical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.