HOXC13, a homeobox-containing gene, is involved in hair development and human leukemia. The regulatory mechanism that drives HOXC13 expression is mostly unknown. Our studies have demonstrated that HOXC13 is transcriptionally activated by the steroid hormone estrogen (17beta-estradiol; E2). The HOXC13 promoter contains several estrogen-response elements (EREs), including ERE1 and ERE2, which are close to the transcription start site, and are associated with E2-mediated activation of HOXC13. Knockdown of the estrogen receptors (ERs) ERalpha and ERbeta suppressed E2-mediated activation of HOXC13. Similarly, knockdown of mixed lineage leukemia histone methylase (MLL)3 suppressed E2-induced activation of HOXC13. MLLs (MLL1-MLL4) were bound to the HOXC13 promoter in an E2-dependent manner. Knockdown of either ERalpha or ERbeta affected the E2-dependent binding of MLLs (MLL1-MLL4) into HOXC13 EREs, suggesting critical roles of ERs in recruiting MLLs in the HOXC13 promoter. Overall, our studies have demonstrated that HOXC13 is transcriptionally regulated by E2 and MLLs, which, in coordination with ERalpha and ERbeta, play critical roles in this process. Although MLLs are known to regulate HOX genes, the roles of MLLs in hormone-mediated regulation of HOX genes are unknown. Herein, we have demonstrated that MLLs are critical players in E2-dependent regulation of the HOX gene.