ABSTRACTAccording to recent studies, long non‐coding RNA X‐inactive specific transcript (XIST) is involved in the development and progression of many malignant tumors including pancreatic cancer. We validated the detailed role of XIST in human pancreatic cancer (PC) cell lines and PC tissues so as to determine its exact function and the mechanism by which it affected PC proliferation. In our research, lncRNA‐XIST was specifically upregulated in PC tissues and cell lines, and high XIST expression in PC was related to poorer prognosis (larger tumor size, perineural invasion, lymph node micrometastases, and shorter overall survival). XIST augmented PC cell proliferation. Recently, the interaction between lncRNA and miRNA has been frequently reported to play major role in several biological processes. In the present study, XIST and miR‐133a reciprocally inhibited each other in PC cells. Exogenous miR‐133a expression significantly inhibited PC cell proliferation. Moreover, as exhibited by luciferase reporter gene assays, miR‐133a bound to XIST and the 3′UTR of EGFR by direct targeting. In PC tissues, miR‐133a expression was down‐regulated and EGFR expression was up‐regulated; miR‐133a was inversely correlated with EGFR and XIST, respectively; XIST was positively correlated with EGFR. Taken together, these findings will shed light on the role and mechanism of XIST/miR‐133a/EGFR in regulating PC cells proliferation. XIST may serve as a potential therapeutic target in PC in the future. J. Cell. Biochem. 118: 3349–3358, 2017. © 2017 Wiley Periodicals, Inc.