We sought to determine whether STAT3 mediated tamoxifen resistance of breast cancer stem cells in vitro. The capacities for mammosphere formation and STAT3 expression of CD44(+)CD24(-/low) MCF-7 and MCF-7 were observed. The CD44(+)CD24(-/low) subpopulation ratio and its sensitivity to adriamycin were analyzed in MCF-7 and TAM resistant (TAM-R) cells. Cell cycle, apoptosis, STAT3 and phospho-STAT3 changes were observed after treatment with tamoxifen. Small interference RNA-mediated knockdown of STAT3 in TAM-R cells was also performed. CD44(+)CD24(-/low) MCF-7 showed higher capacities for mammosphere formation and STAT3 expression than total MCF-7. The CD44(+)CD24(-/low) subpopulation was also upregulated in TAM-R cells with less sensitivity to adriamycin than MCF-7. Cell cycle changes, anti-apoptotic effects and STAT3 changes were also found. Meanwhile, the knock-down of STAT3 in TAM-R resulted in an increase in sensitivity to tamoxifen. It is concluded that STAT3 plays an essential role in breast cancer stem cells, which correlated with tamoxifen resistance.