Combination chemotherapy is being increasingly explored due to its potential superior effectiveness. 10-Hydroxy-2-decenoic acid (10- H2DA), a special fatty acid from royal jelly, has been reported to have antitumor activity. This study investigated antiproliferative effects and underlying mechanisms of 10-H2DA treatments in combination with doxorubicin (DXR), a potent chemotherapeutic compound, against MCF-7 breast cancer cells. By MTS tetrazolium assay, co-treatment of 10-H2DA (0.0125-125 µg/mL) with DXR (1 µM) for 24h significantly inhibited MCF-7 cell proliferation in a variable dose-dependent manner (p<0.05). The highest dose of 125 µg/mL 10-H2DA co-treatment maximally decreased cell proliferation by 71%, whereas DXR treatment alone decreased by 42%, compared to the medium control. Western blot analysis revealed that DXR treatment alone moderately decreased levels of c-MYC (0.3-fold), BCL2/BAX (0.3-fold) and cyclin B1 (0.4-fold) while slightly decreasing CDK4 (0.8-fold); however, it slightly increased cyclin D1 (1.2-fold). In comparison to DXR treatment alone, 125 µg/mL 10-H2DA co-treatment moderately decreased levels of c-MYC (0.4-fold), BCL2/BAX (0.4-fold), cyclin D1 (0.4-fold), and CDK4 (0.3-fold), but extensively increased cyclin B1(3.9-fold). Our research demonstrated 125 µg/mL 10-H2DA co-treatment synergistically enhanced the antiproliferative effects of DXR against MCF-7 breast cancer cells through reduction of c-MYC, the oncoprotein responsible for stimulating cell growth; induction of cell cycle arrest, especially at G0/G1 phases through inhibition of cyclin D1 and CDK4; and induction of apoptosis through inhibition of BCL2/BAX. Further, in vivo studies are required to verify the potential of 10-H2DA for chemotherapy.