Vesicular stomatitis virus (VSV) is a promising vector for vaccine and oncolysis, but it can also produce acute diseases in cattle, horses, and swine characterized by vesiculation and ulceration of the tongue, oral tissues, feet, and teats. In experimental animals (primates, rats, and mice), VSV has been shown to lead to neurotoxicities, such as hind limb paralysis. The virus matrix protein (M) and glycoprotein (G) are both major pathogenic determinants of wild-type VSV and have been the major targets for the production of attenuated strains. Existing strategies for attenuation included: (1) deletion or M51R substitution in the M protein (VSV?M51 or VSVM51R, respectively); (2) truncation of the C-terminus of the G protein (G?28). Despite these mutations, recombinant VSV with mutated M protein is only moderately attenuated in animals, whereas there are no detailed reports to determine the pathogenicity of recombinant VSV with truncated G protein at high dose. Thus, a novel recombinant VSV (VSV?M51-G?28) as well as other attenuated VSVs (VSV?M51, VSV-G?28) were produced to determine their efficacy as vaccine vectors with low pathogenicity. In vitro studies indicated that truncated G protein (G?28) could play a more important role than deletion of M51 (?M51) for attenuation of recombinant VSV. VSV?M51-G?28 was determined to be the most attenuated virus with low pathogenicity in mice, with VSV-G?28 also showing relatively reduced pathogenicity. Further, neutralizing antibodies stimulated by VSV-G?28 proved to be significantly higher than in mice treated with VSV?M51-G?28. In conclusion, among different attenuated VSVs with mutated M and/or G proteins, recombinant VSV with only truncated G protein (VSV-G?28) demonstrated ideal balance between pathogenesis and stimulating a protective immune response. These properties make VSV-G?28 a promising vaccine vector and vaccine candidate for preventing vesicular stomatitis disease.