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Objective This study aimed to investigate how macrophage migration inhibitory factor (MIF) regulates the interaction of signal transducer and activator of transcription 1 (STAT1) with CD74, and affects colon cancer proliferation and invasion.Methods After transfecting MIF small interfering RNA into the SW480 cell line, the expression of STAT1 and CD74 mRNA was detected by qRT-PCR and west blotting. Transwell and MTT assays were performed to detect the colon cancer cell invasion and proliferation ability. Co-immunoprecipitation was used to detect the interaction between CD74 and STAT1 proteins in the treated and control groups. Results The cellular biological assays (MTT and Transwell) showed that the proliferation and invasion ability of colon cancer cells decreased after MIF knockdown; the results showed significant statistical difference (P < 0.05). The results of the co-immunoprecipitation assay suggested that MIF knockdown in colon cancer cells could inhibit the binding of CD74 and STAT1 proteins; statistical difference was observed between the two groups (P < 0.05). Conclusion MIF can increase the proliferation and invasion of colon cancer cells by promoting the combination of CD74 and STAT1.