Stringently controlled in vitro experiments are a necessary part of translational research. Cell lines are useful for exploring the underlying biology of cancer. Very few canine soft tissue sarcoma cell lines exist. This report describes the establishment of a new canine soft tissue sarcoma cell line (MBSa1) derived from a high-grade, metastatic neurofibrosarcoma. The primary tumor tissue was obtained from a 12-year-old neutered male German Shepherd Dog and was maintained in tissue culture for a minimum of 20 passages over 7 months. MBSa1 was injected into athymic mice to determine tumorigenicity. Five million cells were injected into the subcutis of the right flank of athymic nude mice. Nine of the 10 mice grew tumors 1 cm or larger within 8 weeks of cell injection. The large number of in vitro passages coupled with solid tumor formation in athymic nude mice demonstrates that MBSa1 has been immortalized and is tumorigenic.