The matrix (M) protein of vesicular stomatitis virus (VSV) plays significant roles in the replication of VSV through its involvement in the assembly of virus particles as well as by facilitating the evasion of innate host cell defense mechanisms. The presence of methionine at position 51 (M51) of the matrix (M) protein of the VSV Indiana serotype (VSV(Ind)) has been proven to be crucial for cell rounding and inhibition of host cell gene expression. The M protein of VSV(Ind) with the substitution of M51 with arginine (R:M51R) results in the loss of inhibitory effects on host cell gene expression. The VSV(Ind) expressing the M(M51R) protein became the attractive oncolytic virus which is safer and more tumor-specific because the normal cells can clear the mutant VSV(Ind) easily but tumor cells are susceptible to the virus because a variety of tumor cells lack innate antiviral activities. We have studied the role of the methionines at positions 48 and 51 of the M protein of the New Jersey serotype of VSV (VSV(NJ)) in the induction of cytopathic effects (CPE) and host cell gene expression. We have generated human embryonic kidney 293 cell lines inducibly expressing M proteins with M to R mutations at positions 48 and 51, either separately or together as a double mutant, and examined expression of heat shock protein 70 (HSP70) as an indicator of host cell gene expression. We have also generated recombinant VSV(NJ) encoding the mutant M proteins M(M48R) or M(M48R+M51R) for the first time and tested for the expression of HSP70 in infected cells. Our results demonstrated that the M51 of VSV(NJ) M proteins has a major role in cell rounding and in suppressing the host cell gene expression either when the M protein was expressed alone in inducible cell lines or when expressed together with other VSV proteins by the recombinant VSV(NJ). Amino acid residue M48 may also have some role in cell rounding and in the inhibitory effects of VSV(NJ) M, which was demonstrated by the fact that the cell line expressing the double substitution mutant M(M48R+M51R) exhibited the least cytopathic effects and the least inhibitory effect on host cell gene expression.