An outbreak of a canine influenza virus (CIV) H3N2 reassortant derived from pandemic (pdm) H1N1 and CIV H3N2 in companion animals has underscored the urgent need to monitor CIV infections for potential zoonotic transmission of influenza viruses to humans. In this study, we assessed the virulence of a novel CIV H3N2 reassortant, VC378, which was obtained from a dog that was coinfected with pdm H1N1 and CIV H3N2, in ferrets, dogs, and mice. Significantly enhanced virulence of VC378 was demonstrated in mice, although the transmissibility and pathogenicity of VC378 were similar to those of classical H3N2 in ferrets and dogs. This is notable because mice inoculated with an equivalent dose of classical CIV H3N2 showed no clinical signs and no lethality. We found that the PA and NS gene segments of VC378 were introduced from pdmH1N1, and these genes included the amino acid substitutions PA-P224S and NS-I123V, which were previously found to be associated with increased virulence in mice. Thus, we speculate that the natural reassortment between pdm H1N1 and CIV H3N2 can confer virulence and that continuous surveillance is needed to monitor the evolution of CIV in companion animals.