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Abstract Background Expression of human equilibrative nucleoside transporter‐1 (hENT1) is reported to predict survival of gemcitabine (GEM)‐treated patients. However, predictive values of immunohistochemical hENT1 expression may differ according to the antibodies, 10D7G2 and SP120. Aim We aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis. Methods The subjects of this study were totally 332 whose formalin‐fixed paraffin‐embedded specimens and/or unstained sections were obtained. The individual H‐scores and four classifications according to the staining intensity were applied for the evaluation of hENT1 expression by 10D7G2 and SP120, respectively. Results The highest concordance rate (79.8%) was obtained when the cut‐off between high and low hENT1 expression using SP120 was set between moderate and strong. There were no correlations of hENT1 mRNA level with H‐score (p = .258). Although the hENT1 mRNA level was significantly different among four classifications using SP120 (p = .011), there was no linear relationship among them. Multivariate analyses showed that adjuvant GEM was a significant predictor of the patients with low hENT1 expression using either 10D7G2 (Hazard ratio [HR] 2.39, p = .001) or SP120 (HR 1.84, p