Objective To investigate the related mechanism which metformin inhibited the proliferation of HCT116 colorectal cancer cells via down-regulating the expression of up-frameshift protein 1 (UPF1).     Methods TCGA and UALCAN databases were utilized to analyze the expression level of UPF1, while Western blotting and Real-time PCR were performed to validate the differences of UPF1 expressions in colon cancer tissues and adjacent normal tissues. Clone formation assay, CCK-8 assay, wound healing assay and Transwell invasion assay were used to examine the effects of knockdown UPF1 on the proliferation, migration and invasion of HCT116 cells respectively. The HCT116 cell dataset with UPF1 knockdown was screened from GEO database for Kyoto Encydopedia of Genes and Genomes(KEGG) pathway analysis, the expression level of differential genes that enriched in Hippo pathway were verified by Real-time PCR. The HCT116 cells were treated with metformin, Western blotting and Real-time PCR were employed to detect the UPF1 expression. Mendelian randomization analysis was performed to explore the causal association between metformin treatment and colorectal cancer.     Results Analysis of TCGA and UALCAN databases showed that both UPF1 mRNA and protein were highly expressed in colon cancer tissues and the expression level of UPF1 was closely correlated with clinicopathologic stage and lymph node metastasis. Compared with adjacent normal tissues, the UPF1 protein and mRNA were highly expressed in colon cancer tissues. Knockdown UPF1 expression could inhibit the proliferation, migration and invasive ability of HCT116 cells. There were 8 differential genes affect the Hippo pathway by KEGG enrichment analysis, Real-time PCR experiments confirmed that CTNNB1, BMP4, TEAD2, PARD6G and FZD1 mRNA decreased in HCT116 cells with UPF1 knockdown. Both UPF1 protein and mRNA expressions decreased after metformin treatment in HCT116 cells. Mendelian randomization analysis showed a negative causal association between metformin treatment and colorectal cancer.      Conclusion Knockdown of UPF1 expression inhibits the proliferation of HCT116 cells through regulating Hippo pathway. Metformin can reduce the UPF1 expression for further inhibiting the proliferation of colorectal cancer cells.