Although it is known that STAT3 transcriptional activity is modulated by phosphorylation at serine residue 727, the role of STAT3 serine phosphorylation in breast cancer remains mostly unexplored. In this study, we examined the expression patterns of serine residue 727-phosphorylated STAT3 (p-ser727-STAT3) in breast infiltrating ductal carcinoma tissues and nearby noncancer tissues by using immunoblotting techniques, and correlated the expression profiles with clinicopathological characteristics. A significantly elevated p-ser727-STAT3 expression was observed in 61.8% (42/68) of breast cancer tissues as compared to corresponding noncancer tissues (p < 0.001). Further, immunohistochemical analysis also showed an increased nuclear p-ser727-STAT3 staining in cancer lesions. The increased p-ser727-STAT3 expression in breast infiltrating ductal carcinoma tissues correlated significantly with negative estrogen receptor (ER) status, increased stage of cancer and increased tumor size (p = 0.001, 0.024 and 0.014, individually). Intriguingly, we noticed that the expression levels of p-ser727-STAT3 in ER-negative breast cancer cell lines were higher than those in ER-positive breast cancer cell lines. In ER-positive MCF7 cells, treatment with ERalpha-specific siRNA increased, whereas treatment with anticancer drug tamoxifen decreased the expression of p-ser727-STAT3, phenomena not observed in ER-negative MDA-MB-231 cells. In conclusion, our results suggest that p-ser727-STAT3 may be involved in the pathogenesis of breast cancer in an ER-dependent manner.