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Cancer stem‐like cells (CSLCs) in solid tumors are thought to be resistant to conventional chemotherapy or molecular targeting therapy and to contribute to cancer recurrence and metastasis. In this study, we aimed to identify a biomarker of pancreatic CSLCs (P‐CSLCs). A P‐CSLC‐enriched population was generated from pancreatic cancer cell lines using our previously reported method and its protein expression profile was compared with that of parental cells by 2‐D electrophoresis and tandem mass spectrometry. The results indicated that a chaperone protein calreticulin (CRT) was significantly upregulated in P‐CSLCs compared to parental cells. Flow cytometry analysis indicated that CRT was mostly localized to the surface of P‐CSLCs and did not correlate with the levels of CD44v9, another P‐CSLC biomarker. Furthermore, the side population in the CRT high /CD44v9 low  population was much higher than that in the CRT low /CD44v9 high  population. Calreticulin expression was also assessed by immunohistochemistry in pancreatic cancer tissues ( n  = 80) obtained after radical resection and was found to be associated with patients' clinicopathological features and disease outcomes in the Cox proportional hazard regression model. Multivariate analysis identified CRT as an independent prognostic factor for pancreatic cancer patients, along with age and postoperative therapy. Our results suggest that CRT can serve as a biomarker of P‐CSLCs and a prognostic factor associated with poorer survival of pancreatic cancer patients. This novel biomarker can be considered as a therapeutic target for cancer immunotherapy.

Calreticulin is highly expressed in pancreatic cancer stem‐like cells