In the present study, 13 clinical cases of canine mammary adenocarcinoma were evaluated in order to understand the effect of Tarantula cubensis extract (TOE) on tumor tissue. Punch biopsies were taken from the tumors before treatment with TOE. Subcutaneous injections of TOE were administered three times at weekly intervals (3 mL per dog). Between days 7 and 10 after the third injection, the tumor masses were extirpated by complete unilateral mastectomy. Pre- and post-treatment tumor tissues were immunohistochemically assessed. The expression of B-cell lymphoma 2 (Bcl.2) was found to be higher in pre-treatment compared to post-treatment tissues (p <0.01) whereas Ki-67 expression was lower in post-treatment tissues (p < 0.01). No significant differences in fibroblast growth factor or vascular endothelial growth factor expression were observed between pre- and post-treatment tissues (p > 0.05). The apoptotic index was determined to be low before treatment and increased during treatment. These results suggest that TOE may be effective for controlling the local growth of canine mammary adenocarcinoma by regulating apoptosis.