BackgroundThe prognosis of distal cholangiocarcinoma (dCCA) remains poor; thus, the identification of new therapeutic targets is warranted. Phosphorylated S6 ribosomal protein indicates a mammalian target of rapamycin complex 1 (mTORC1) activity, and mTORC1 plays a central role in controlling cell growth and regulating glucose metabolism. We aimed to clarify the effect of S6 phosphorylation on tumor progression and the glucose metabolic pathway in dCCA.MethodsThirty-nine patients with dCCA who underwent curative resection were enrolled in this study. S6 phosphorylation and the expression of GLUT1 were evaluated by immunohistochemistry, and their relationship with clinical factors was investigated. The effect of S6 phosphorylation on glucose metabolism with PF-04691502 treatment, an inhibitor of S6 phosphorylation, was examined in cancer cell lines by Western blotting and metabolomics analysis. Cell proliferation assays were performed with PF-04691502.ResultsS6 phosphorylation and the expression of GLUT1 were significantly higher in patients with an advanced pathological stage. Significant correlations between GLUT1 expression, S6 phosphorylation, and SUV-max of FDG-PET were shown. In addition, cell lines with high S6 phosphorylation levels showed high GLUT1 levels, and the inhibition of S6 phosphorylation reduced the expression of GLUT1 on Western blotting. Metabolic analysis revealed that inhibition of S6 phosphorylation suppressed pathways of glycolysis and the TCA cycle in cell lines, and then, cell proliferation was effectively reduced by PF-04691502.ConclusionUpregulation of glucose metabolism via phosphorylation of S6 ribosomal protein appeared to play a role in tumor progression in dCCA. mTORC1 may be a therapeutic target for dCCA.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12876-023-02815-2.