In addition, the inhibitory effect of gemcitabine on PGK1 enzymatic activity and cell proliferation was validated through in vitro biochemistry assay, cultured ESCC cells, and xenografts model, in which the levels of PGK1 phosphorylation, but not PGK1 protein levels, were positively associated with tumors weight. Here, PGK1 is linked to esophageal squamous cell carcinoma.