Foxi1, a forkhead family of transcription factor, in narrow and clear cells in epididymis is required for male fertility through regulating transcription of vacuolar H+-ATPase. To understand the regulation of Foxi1 gene activation in epididymis, the effects of steroids and their receptor antagonists and testicular factors on the expression of Foxi1 in epididymal segments were examined in mouse. Epididymis were sampled from adult mice following injections of ICI 182,780 (5mg/head, 2 times for 15 days), dexamethasone (DEX, 0.1,1,10ug/kg/day for 5 days) or oral administration of flutamide (FLM, 100mg/kg/day for 10 days). Otherwise, adult mice were orchidectomized (ORX), rested for 2 weeks, and received testosterone propionate(TP, 3mg/kg/day) for 7 days. In addition, adult male mice were subjected to efferent duct ligation (EDL) and epididymis was collected after 15 days. To study estrogen regulation of Foxi1 gene activation via estrogen receptor α (ESR1), Foxi1 expression was examined in ESR1 knock-out mice epididymis. Expression and subcellular localization of Foxi1 was analyzed by realtime RT-PCR and immunohistochemistry. To search transcription factor binding in the mouse Foxi1 gene promoter, in silico analysis was performed using TESS, TFSEARCH, and Gene-Regulation. ICI 182,780 significantly decreased Foxi1 mRNA levels in caput and corpus but increased in cauda epididymis. Foxi1 mRNA levels in caput epididymis of ESR1 KO mice were significantly lower than those of WT mice, but no significantly changed in corpus and cauda epididymis. Taken together, estrogen differentially regulates Foxi1 gene expression in epididymis. In ORX mice, Foxi1 mRNA levels were significantly increased in epididymis, and which was abrogated by TP. Though FLM did not significantly alter the Foxi1 mRNA levels, androgen may affect Foxi1 gene expression in epididymis. DEX significantly decreased Foxi1 mRNA levels in caput and corpus epididymis at 0.1ug/kg/day and in cauda epididymis at 1ug/kg/day, suggesting that glucocorticoid may negatively regulate Foxi1 gene expression. No significant change in Foxi1 mRNA levels was found after EDL. Foxi1 immunoreactivity was found in the nuclei of narrow cells of caput epididymis including initial segment and clear cells of corpus and cauda epididymis. Of note, in ORX mice, Foxi1-positive narrow cells and clear cells were increased, and which was abrogated by TP. In silico analysis revealed the presence of putative binding sequences for ESR1, AR, and GR in the 5’ upstream region from the Foxi1 promoter. In conclusion, the expression of Foxi1 in narrow cells in caput epididymis might be positively regulated by estrogen via ESR1, which was different from estrogen–ESR signaling in clear cells in corpus and cauda epdididymis. Androgen and glucocorticoid may negatively regulate expression of Foxi1 in all epdididymial segments.