Vesicular stomatitis virus (VSV) can cause serious disease and economic loss in live stock animals. No suitable vaccine has been available to prevent it. Although matrix protein (M) has been proved as a virulent factor in rodents, its role in pathogenesis of live stock animals was still unknown. Pig is one of the natural hosts of VSV. However, as the promising vaccine vector candidate, safety and efficacy of M mutant VSV has not been assessed in it. To address the issue and develop VSV vector targeted at M for swine, recombinant viruses with different mutated M proteins were evaluated. Due to the existing virulence of VSV with M51 deletion (VSVΔM51) in pig, a novel recombinant one with triple mutations in M combined with M51 deletion, V221F, S226R (VSV-MT) was constructed, which proved significantly attenuated. Based on the in vivo and in vitro studies, it was proved that M protein is truly a virulent factor for VSV in pig and can be an ideal target to develop safe and effective vaccine vectors for swine.