Poliovirus oncolytic immunotherapy is a putatively novel approach to treat pediatric brain tumors. This work sought to determine expression of the poliovirus receptor (PVR), CD155, in low-grade and malignant pediatric brain tumors and its ability to infect, propagate, and inhibit cell proliferation. CD155 expression in pleomorphic xanthoastrocytoma (PXA), medulloblastoma, atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, and anaplastic ependymoma specimens was assessed. The ability of the polio: rhinovirus recombinant, PVSRIPO, to infect PXA (645 [BRAF V600E mutation], 2363) and medulloblastoma (D283, D341) cells were determined by viral propagation measurement and cell proliferation. PVR mRNA expression was evaluated in 763 medulloblastoma and 1231 normal brain samples. CD155 was expressed in all 12 patient specimens and in PXA and medulloblastoma cell lines. One-step growth curves at a multiplicity of infection of 10 demonstrated productive infection and peak plaque formation units at 5–10 hours. PVSRIPO infection significantly decreased cellular proliferation in 2363, 645, and D341 cell lines at 48 hours (p < 0.05) and resulted in cell death. PVR expression was highest in medulloblastoma subtypes Group 3γ, WNTα, and WNTβ (p < 0.001). This proof-of-concept in vitro study demonstrates that PVSRIPO is capable of infecting, propagating, prohibiting cell proliferation, and killing PXA and Group 3 medulloblastoma.