We here investigate the effect and mechanism by which UCHL1 regulates breast cancer chemotherapy resistance by targeting drug efflux and breast cancer stem cells(BCSC). Expression of UCHL1 in adriamycin-resistant human breast cancer cells(MCF-7/ADM) and the parental line MCF-7 were compared. Using LDN57444 inhibiting UCHL1 function, we detected half maximal inhibitory concentration(IC50) of MCF-7/ADM cells, ADM accumulation and CD44⁺CD24^- BCSC in MCF-7/ADM cells by FACS, the microsphere formation ability of MCF-7/ADM, and expression of drug efflux pump protein and stem cell related genes by real-time PCR and western blotting. We showed that UCHL1 expression was substantially higher in MCF-7/ADM cells than in MCF-7 cells. After UCHL1 inhibition in MCF-7/ADM, IC50 of these cells decreased significantly, drug efflux pump protein P-gp and MRP1 decreased and intracellular drug accumulation increased accordingly. What's more, the percentage of CD44⁺CD24^- BCSC, the ability of microsphere formation and stem cell related gene expression were also decreased. These results suggest that UCHL1 can promote chemotherapy resistance of breast cancer cells, mainly through drug efflux pump protein-dependent classical mechanism in chemotherapy resistance, but also closely related to promoting BCSC.