HER2 positive breast cancer is one of the biggest health problems in the world, causing millions of deaths every year. Drug combination modeling studies are extensively evaluated in treating many diseases. Pharmacological studies over the last half-century have shown that gambogic acid has potent anti-tumor activity against many types of cancer, including breast cancer. In this study, we examined the synergistic anticancer effect of gambogic acid and trastuzumab in HER2 positive breast cancer cell line (MDA-MB-453). In-vitro synergistic and antiproliferative effects of trastuzumab plus gambogic acid studies were determined with XTT method and the combination index (CI) values of the trastuzumab and gambogic acid combination were calculated by CompuSyn software. To determine molecular mechanisms of the trastuzumab and gambogic acid combination in MDA-MB-453 cells, the differences of gene and protein expression levels of HER2, caspase-9 and Bax were analyzed with using RT-qPCR and ELISA techniques. The combination of 50 μg/ml trastuzumab and 5 μM gambogic acid showed the best synergistic effect at 24 h incubation in MDA-MB-453 cells according to the in-vitro cell proliferation, RT-qPCR and ELISA test. Gambogic acid effects on HER2 positive breast cancer cell line shows its potential as natural compound to inhibit breast cancer cell proliferation in combination with trastuzumab.