Background: Ulcerative colitis (UC) is a prevalent case which needs more detailed exploration. We attempted to illustrate how miR-223-3p acted in the development of UC. Materials and Methods: The level of miR-223-3p in serum from UC patients was detected using quantitative reverse-transcriptase polymerase chain reaction. The in vivo experimental spleen-kidney-yang deficiency mice model was established to get a better understanding of the action of miR-223-3p in UC development. Hematoxylin and eosin staining was applied to observe the intestinal epithelial barrier function. Western blotting was applied to measure the expression of ZO-1, Occludin, TGF-β, and Smad3. Results: Enrichment of miR-223-3p was observed in serum from UC patients. miR-223-3p antagomir could lower the shortening of colon length, alleviating the intestinal epithelial barrier function damage caused by UC. Under the UC conditions, TNF-α, IL-6, and IL-1β were up-regulated, whereas with the miR-223-3p antagomir, the up-regulation was less pronounced; ZO-1 and Occludin protein levels were decreased, whereas the decrease was less obvious in the miR-223-3p antagomir group. Additionally, miR-223-3p regulated UC development through the TGF-β/Smad3 pathway. Conclusion: miR-223-3p promoted UC development with spleen-kidney-yang deficiency through the TGF-β/Smad3 pathway.