Ulcerative colitis (UC), with high morbidity has become one of the fastest-growing severe illnesses in the world. Although MiR-29a is highly expressed in the tissues of UC patients, the mechanism of miR-29a involved in the specific pathogenesis of UC is not known. In this study, a GFP-light chain 3 (LC3) immunofluorescence assay was used to observe the formation of the autophagic spot; qRT-PCR and western blotting analyses were carried out to detect the expression of autophagy-related proteins, including BECN1, Autophagy-related gene (ATG)5, ATG16L, and transcription factor EB. The dual-fluorescence reporter assay was used to analyze the direct effect of miR-29a on ATG9A; experimental dextran sulfate sodium-induced colitis in mice was used to establish the UC model. Our studies showed that the overexpression of miR-29a not only suppressed the production of GFP-LC3 autophagy spots but also inhibited the level of LC3II/LC3I and upregulated the expression of P62 in HT29 and HCT116 cells. Moreover, the results showed that miR-29a directly targeted the 3′UTR region of ATG9A mRNA to suppress the activation of HT29 and HCT116 cells’ autophagy. Also, overexpression of ATG9A rescued rapamycin-induced autophagy that was inhibited by overexpression of miR-29a. In addition, miR-29a also affected the expression of autophagy-related proteins (BECN1, ATG5, ATG16L1, and transcription factor EB). Notably, miR-29a was upregulated, whereas ATG9A was downregulated in the experimental dextran sulfate sodium-induced colitis in mice. In effect, this study showed that miR-29a inhibits rapamycin-induced intestinal epithelial cells’ autophagy partly by decreasing ATG9A in UC. These findings may provide new insights that may help control the inflammation in UC.