Multiple injections of bone marrow mesenchymal stem cells (BMMSCs) have been used for treatment of chronic colitis in mice. We aimed to report the therapeutic effects of a single injection of human umbilical cord mesenchymal stem cells (hUCMSCs) on acute and chronic colitis. Male C57BL/6JNarl mice were divided into control, phosphate-buffered saline (PBS), and hUCMSCs treated groups, respectively. Acute and chronic colitis were induced in the mice (except controls) using 3% dextran sulfate sodium (DSS). The mice in the hUCMSCs group underwent a single injection of hUCMSCs. The disease activity index (DAI), colon length, histology, colon inflammation score, in vivo stem cells images, and blood cytokine levels were recorded. The DAI was significantly higher in the hUCMSCs group than in the control group and lower than in the PBS group on all days. The colon length was significantly longer and the colon inflammation score was significantly lower in the hUCMSCs group than in the PBS group on days 8 and 25. IL17A, Gro-α, MIP-1α, MIP-2, and eotaxin were significantly lower in the hUCMSCs group than in the PBS group on days 8 and 25. Single-injection hUCMSCs improved DSS-induced acute colitis and decreased progression of acute colitis to chronic colitis.