Canine mammary tumors are the most common neoplasms in intact female dogs. The surgery cannot always solve the problem, chemotherapy are recommend to these patients. However, chemotherapy could always fail because of multidrug resistance (MDR). Through stepwise increasing 5-Fluorouracil (5-FU) concentration in the culture medium, a 5-FU-resistant canine mammary tumor cell line CMT7364/5-FU was established to disclose the molecular mechanism of the drug resistance. Cell morphology, cell sensitivity to drugs, growth curves, expression of proteins, and chemo-sensitivity in vivo were compared between the parental cell line and resistant cell line. As compared it to its parental cell line (CMT7364), CMT7364/5-FU showed different morphology, cross-resistant to other chemo-drugs and a prolonged population doubling time (PDT). The drug efflux pump proteins (ABCB1 and ABCG2) in CMT7364/5-FU were up-regulated. In vivo, the similar result revealed that CMT7364/5-FU cell line was more resistant to 5-FU. In conclusion, a 5-FU-resistant canine mammary tumor cell line (CMT7364/5-FU) was successfully established, it can serve as a good model for researching the mechanism of MDR and screening effective agents to reverse drug resistance.