Background: Oncolytic virotherapy can serve as a novel therapeutic strategy in oncology. In this study, we aimed to evaluate the oncolytic activity of the mumps virus RS-12 strain after its adaptation to cancer cells via serial passaging. Methods: To adapt the RS-12 strain-based vaccine to cancer cells, it was passaged eight times in the HT1080 cell line and was isolated via two terminal endpoint dilutions. The genetic homogeneity of isolated cancer cell-adapted RS-12 variant was confirmed by direct sequencing of regions, encompassing four known heterogeneous genomic positions. The in vitro cytotoxic effects of viruses was assessed in two different cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptosis-inducing effects of the cancer cell-adapted variant and its parental virus on cancer cells were quantified by flow cytometry. Results: According to the chromatograms, the RS-12 strain vaccine seed exhibited two peaks at the genomic nucleotide positions 1591, 2417, 3774, and 12977. On the contrary, cancer cell-adapted RS-12, isolated by terminal endpoint serial dilutions, contained no viral subpopulations in these positions. A significant improvement was observed in the oncolytic potency of our cancer cell-adapted variant compared to its parental virus vaccine seed in vitro. Besides, the variant efficiently induced apoptosis in the human fibrosarcoma and adenocarcinoma cell lines. Conclusions: Considering the increased oncolytic potency and apoptosis-inducing capacity of this variant in cancer cells, it can be a promising option for future experiments.