Tumors in cattle are uncommon, partly due to their relatively shorter lifespan, and vaginal neoplasms are rarely documented. Among vaginal and vulvar tumors, fibropapillomas are the most common, while leiomyomas, leiomyosarcomas, fibrosarcomas, and fibroleiomyosarcomas occur far less frequently. We describe the clinical presentation, surgical management, and pathological characterization of a vaginal fibroleiomyosarcoma in a 5-year-old Holstein cow. Clinical examination revealed a firm intravaginal mass attached to the dorsal wall. Surgical excision was performed under epidural anesthesia, followed by appropriate post-operative antibiotic and analgesic therapy. The cow recovered without complication, and no recurrence was observed during six months of follow-up. Macroscopically, the tumor measured 65.00 × 52.00 × 40.00 mm, and it was firm in consistency and gray in color. Histopathologically, it was composed of spindle-shaped neoplastic cells arranged in fascicles and bundles, displaying marked pleomorphism, blunt-ended nuclei, eosinophilic cytoplasm, and numerous mitotic figures. The stromal component showed variable collagen deposition, giving the lesion a biphasic fibromatous and leiomyomatous pattern. Immunohistochemically, neoplastic cells showed strong cytoplasmic positivity for α-smooth muscle actin and vimentin, confirming both smooth muscle and mesenchymal differentiation. Based on these findings, the tumor was diagnosed as a primary vaginal fibroleiomyosarcoma. To the best of our knowledge, this represents the second report of such a tumor in cattle and the first described in a Holstein cow. The case highlights the diagnostic value of combining histopathological and immunohistochemical analyses for accurate identification of rare bovine vaginal tumors.