The aim of this study was to establish a murine protothecal mastitis model and to evaluate the treatment efficiency of gentamicin. Challenge routes were determined with a pathogenic Prototheca zopfii genotype 2 (P. zopfii) strain. 25 BALB/c mice were inoculated in mammary glands with graded dosages (10(3), 10(4), 10(5), 10(6), 10(7) CFU of P. zopfii) and killed on the 7th day. Another 25 animals were also killed at 1, 3, 5, 7, and 9 days after inoculation of 1 × 10(6) CFU of P. zopfii, the milk somatic cell counts, pathological section of mammary glands, and P. zopfii burden were observed. The antimicrobial activity was tested using disc diffusion test and minimum inhibitory concentrations. Gentamicin was given intramuscularly to analyze the therapeutic effect. The results showed that the best infection route was intra-mammary gland, and the mastitis model was established with 1 × 10(6) CFU of P. zopfii. After infection, the somatic cell counts increased significantly. The pathological reaction mainly consisted of infiltration of inflammatory cells, destruction of acini, accumulation of lymphocyte cells and the severity of the changes was dosage and time-dependent. The P. zopfii burden revealed that P. zopfii continuously replicated. In vitro susceptibility tests indicated that the Prototheca strains were antimicrobial susceptible to gentamicin at concentrations between 0.03 and 4 μg/ml. In vivo therapeutic assay demonstrated that high concentrations of gentamicin (≥20 mg/kg) could inhibit the growth of P. zopfii. We conclude that the murine model of protothecal mastitis was established successfully and gentamicin may be an effective choice for treatment of P. zopfii.