Objective Observe the effect of microRNA-574-3p (miR-574-3p) on the proliferation of colon cancer cells, and explore its potential mechanism. Methods HCT116 colon cancer cells were transfected with miR-574-3p mimics or a negative control and used miR-574-3p mimics and negative control groups, respectively. Normal HCT116 cells were used as the blank control group. RT-qPCR was used to detect expression of miR-574-3p. CCK-8 assays were used to measure cell proliferation. Colony formation assay was used to assess cell clone formation. Flow cytometry was used to detect cell cycle changes. Western blot was used to detect expression of Cyclin A1, CDK2, PCNA, Cdc25A, and p53 proteins. Results Compared with the negative control group, expression of miR-574-3p in the miR-574-3p mimics group was upregulated significantly (P<0. 01), and cell viability was significantly reduced at 36 ~ 72 h (P<0. 01). Compared with the negative control group, the colony formation rate was significantly reduced in the miR-574-3p mimics group (P<0. 01), while the proportion of S-phase cells was increased significantly (P< 0. 01). Compared with the negative control group, expression of Cyclin A1, CDK2, PCNA, and Cdc25A was significantly downregulated in the miR-574-3p mimic group (P< 0. 01) and expression of p53 protein was upregulated significantly (P<0. 01). There was no significant difference in the above indicators between negative control and blank control groups. Conclusions MiR-574-3p inhibits the proliferation of HCT116 colon cancer cells, which may be related to the downregulation of Cyclin A1, CDK2, PCNA, and Cdc25A and the upregulation of p53 protein expression, which ultimately induces cell cycle arrest in S phase.