Objective To observe the effect of miR-424-5P on Src-YAP signaling and the proliferation of colon cancer cells. Methods Colon cancer HT-29 cells were used. Exogenous up-regulation of miR-424-5P was achieved in HT- 29 cells by transfection and a blank group (Blank group), an empty vector group (NC group) and an overexpression group (miR-424-5P-OV group) were prepared. RTFQ-PCR was used to detect the level of miR-424-5P. MTT assays were used to detect cell viability. The nuclear distribution of YAP was detected by immunofluorescence. Western blot was used to detect the levels of Src, YAP, LATS1 and their phosphorylation. Results Compared with the NC group, the miR-424-5P RNA level in the miR-424-5P-OV group was significantly increased (P <0.01). The overexpression of miR-424-5P significantly reduced the viability and colony-forming ability of HT-29 cells (P <0.01). In the blank and NC groups, YAP was mainly localized in the nucleus, with little cytoplasmic distribution, while in the miR-424-5P-OV group, YAP showed obvious cytoplasmic and nuclear distribution. Overexpression of miR-424-5P significantly up-regulated p-YAP and p-LATS1 protein levels (P <0.01) and down-regulated p-Src protein levels (P <0.01). Conclusions Overexpression of miR-424-5P can inhibit the proliferation of colon cancer HT-29 cells, which may be related to weakened Src-YAP signaling.