Despite the application of H-1 parvovirus as an anticancer drug, the relationship between its specific tropism and oncolytic activity has been unknown. H-1 viral infection induced cytopathic effects in HeLa cells, whereas Kilham rat virus (KRV), similar to H-1 virus, did not. To explore which segments of the viral protein 2 (VP2) capsid protein in the H-1 virus determine susceptibility to human cancer cells, chimeric H-1 viruses with specific gene segments of H-1 VP2 were constructed. Delineation of the VP2 capsid protein revealed a minimum domain (K208–L435 in the H-1 VP2 protein) to determine infectivity in human cancer cells; however, this domain was not sufficient to maintain infectivity. To solve this problem, further construction of chimeric H-1 viruses illustrated the necessity of segments covering both M1-N87 and D104-P206 in the H-1 VP2 protein, based on chimeric H-1 viruses designated as YCH44, YCH45, and YCH46. Both the variable region 4b (VR4b) domains from KRV VP2 and VR8 from H-1 VP2 were required for the same purpose, based on chimeric H-1 viruses designated as YCH-HK8, YCH16, YCH17, YCH18, and YCH19. We confirmed that chimeric viruses carrying these segments infected human lung adenocarcinoma A549 and pancreatic cancer Panc-1 cells, whereas the parental KRV did not. Taken together, these findings indicate that specific domains of the H-1 virus VP2 capsid protein determine infectivity toward human cancer cells.