Estrogen receptor (ER) α is essential for estrogen-dependent growth, and its level of expression is a crucial determinant of response to endocrine therapy and prognosis in ERα-positive breast cancer. Breast cancer patients show a wide range of ERα expression levels and the levels of expression in individual patients change during disease progression and in response to systemic therapies. However, little is known about how the expression of ERα in human breast cancer is regulated. Recently, several microRNAs (miRNAs) that directly target ERα have been identified, and we previously demonstrated that miR-206 expression was downregulated in ERα-positive human breast cancer. In this study, expression levels of miRNAs that directly target ERα, including miR-18a, miR-18b, miR-22, miR-193b, miR-221/222 and miR-302c, were analyzed in human breast cancer samples by quantitative reverse transcription-PCR analysis. Correlations between the expression levels of these miRNAs and clinicopathological factors, including prognosis, were analyzed. miR-18a expression was much higher in ERα-negative than in ERα-positive tumors (P < 0.0001), with the expression levels of miR-18a not differing in ERα-positive breast cancer as a function of ERα protein level. Surprisingly, the expression levels of miR-193b and miR-221 were significantly lower in ERα-negative than in ERα-positive tumors (P = 0.0015 and P = 0.0045, respectively), and the levels of these miRNAs gradually increased as ERα protein expression increased. There was no statistically significant association between miR-22 and ERα expression, and miR-302c expression was minimal in human breast cancer samples. Prognostic analysis showed that low miR-18b expression was significantly associated with improved survival in HER2-negative breast cancer, although miR-18b expression was not correlated with ERα protein expression. Our results suggest that miRNAs that directly target ERα have distinct roles in not only regulating ERα but also regulating other target genes in human breast cancer.