Abstract African swine fever continues to pose a global agricultural problem due to the absence of vaccine prevention and the high cost of anti-epizootic measures. This study examines the functional role of the African swine fever virus (ASFV) 1L-5-6L multigene family 110 genes in vivo. Four clinically healthy Large White pigs were used in this study. Two groups of animals were inoculated with either the parental strain or the deletion variant, respectively. For subsequent challenge infection, the homologous virulent strain “Stavropol 01/08” was used. Blood samples were collected at specific time intervals. The ASFV infectious activity was determined by titration in porcine blood-derived macrophages. Virus-specific antibodies to the ASFV p30 protein were detected using an enzyme-linked immunosorbent assay (ELISA). The results of real-time polymerase chain reaction (PCR) showed a significant difference in Ct values between samples from the two groups of animals. The determination of ASFV infectious activity in blood samples demonstrated the presence of the virus in animals immunized with the parental strain. The virus was not detected in samples from animals immunized with the deletion strain. The ELISA method demonstrated the presence of p30 protein antibodies in serum samples from 10 to 14 days after immunization with the parental strain, while no antibodies were detected in serum samples from animals immunized with the deletion strain. The properties of the ASFV recombinant strain “Volgograd/D(1L-5-6L) MGF110” were studied in an in vivo experiment. It was found that the deletion strain does not reproduce in animals, unlike the parental strain.