Diffuse gliomas are incurable, prevalent, and aggressive central nervous system tumors. Therefore, the development of selective oncolytic viral strains for malignant neoplasms is highly relevant. This study aimed to create an oncolytic virus based on a vaccine strain of poliovirus type 3 with natural antitumor activity. To achieve this goal, we replaced the internal ribosome entry site (IRES) of poliovirus with the corresponding fragment of human rhinovirus 30. The resulting recombinant oncolytic strain RVP3 retained the serotype of poliovirus type 3, as confirmed by virus neutralization micro-test with specific antiserum. In addition, the oncolytic efficacy of RVP3 was assessed in vitro on a broad panel of cell cultures. According to the results, RVP3 has changed its tropism, losing the ability to replicate in conditionally normal cell lines of embryonic astrocytes and embryonic fibroblasts while retaining the ability to replicate in tumor cells.