Background and Purpose: Breast cancer is the leading cause of death in women worldwide, with resistance to current therapeutic strategies, including tamoxifen, causing major clinical challenges and leading to more aggressive and metastatic disease. To address this, novel strategies that can inhibit the mechanisms responsible for tamoxifen resistance need to be assessed. Experimental Approach: We examined the effect of the novel, clinically-trialled, thiosemicarbazone anti-cancer agent, DpC, and its potential as a combination therapy with the clinically used estrogen receptor (ER) antagonist, tamoxifen, using both tamoxifen-resistant and -sensitive, human breast cancer cells (MDA-MB-453, MDA-MB-231 and MCF-7) in 2D and 3D cell-culture. Synergy was assessed using the Chou-Talalay method. The molecular and anti-proliferative effects of these agents and their combination was examined via Western blot, immunofluorescence and colony formation assays. Key Results: Combinations of tamoxifen with DpC were highly synergistic, leading to potent inhibition of cell proliferation, colony formation, and ER-α transcriptional activity. The combination also more efficiently reduced major molecular drivers of proliferation of tamoxifen-resistant cells, including c-Myc, cyclin D1, and p-AKT, while up-regulating the cell cycle inhibitor, p27, and inhibiting oncogenic phosphorylation of ER-α at Ser167. Assessing these effects using 3D cell culture further confirmed the greater effects of DpC combined with tamoxifen in reducing ER-α expression, and that of the proliferation marker, Ki-67, in both tamoxifen-sensitive and -resistant MCF-7 spheroids. Conclusions and Implications: These studies demonstrate that the synergistic combination of DpC with tamoxifen could be a promising new therapeutic strategy to overcome tamoxifen resistance in ER-positive breast cancer.