HER2 signaling to Rac1 GTPase and Rac1 pathway deregulation have been associated to increased survival of HER2-overexpressing breast cancer cells and resistance to targeted therapy. In this scenario, we evaluated the effects of Rac1 inhibition on breast cancer cells treated with trastuzumab, a HER2-directed antibody widely used for patient treatment. First, we assessed the effect of trastuzumab and the novel Rac1 inhibitor 1A-116 on the viability of BT-474 and SK-BR-3 cell monolayers. Not only single drug treatment significantly decrease the viability of both cell lines (p<0.05), but also a synergistic interaction was found using Combenefit software (p<0.05). To further study this effect, we evaluated drug combinations in 3D cultures. Whereas the growth of BT-474 cell spheroids was inhibited by both trastuzumab and 1A-116 (p<0.05), drug combination induced a significant reduction in spheroid volume (p<0.05). Moreover, interesting results were seen using trastuzumab-resistant BT-474-R cells. Spheroid growth was diminished by trastuzumab and 1A-116 separately compared to control spheroids (p<0.05). However, a significant reduction in spheroid volume was found after treatment with trastuzumab in combination with 1A-116 (p<0.05). To address the cellular processes behind Rac1 inhibition, we analyzed proteomic profiling data. Through a mass spectrometry-based label-free quantification approach, BT-474 cells treated with trastuzumab and 1A-116 were compared with cells treated with trastuzumab alone. The deregulation of proteins governing G1/S transition appeared to be particularly relevant (p<0.05). Thus, we next studied cyclin D1 expression and the cell cycle. We found significantly lower levels of cyclin D1 in BT-474 cells treated with both drugs together (p<0.05), along with an upward trend in G0/G1 cell cycle arrest. These results push us forward to further investigate the potential benefits of targeting Rac1 pathway in breast cancer patients with HER2-overexpression.