Objective To investigate the effects of vitamin D₃ (VitD₃) on the colitis-associated colorectal cancer (CAC) based on the signal pathway of Janus kinase 2 (JAK2)/signal transduction and transcription activator of transcription 3 (STAT3).Methods Thirty male Kunming mice were given dextran sodium sulfate (DSS) and randomly divided into model group, VitD₃ low-dose and high-dose groups, and 10 normal mice were used as control group. The mice in VitD₃ low-dose and high-dose groups were respectively given 10 IU·g^-1 and 20 IU·g^-1 of VitD₃ by gavage at a dose of 2 mL for 28 consecutive days. The mice in the control group and model group were given the same volume of peanut oil. The weight of mice was weighed on the 7th, 14th, 21st, and 28th day of modeling. After the modeling, the colon tissue was removed and the number of tumors was calculated. HE staining, ELISA, and immunohistochemistry were used to detect the colonic tissue morphology, the content of inflammatory factors in the tissue homogenate supernatant, and the expression of epithelial cadherin (E-cadherin) and vimentin. The expressions of H19, let-7a, c-Myc mRNA and HMGA2 mRNA were detected by RT-qPCR. The protein expression of JAK2/STAT3 pathway was detected by Western blotting.Results Compared with the control group, the weight of mice in the model group was significantly reduced at each time point, and there was tumor in the colon. The expression levels of H19, c-Myc mRNA, HMGA2 mRNA, p-JAK2 and p-STAT3 protein, and the score of vimentin protein in colon tissue all were significantly increased, and so were the serum TNF-α, IL-1β, IL-6 expression levels (P<0.05). The let-7a expression level, E-cadherin protein score, and serum IL-10 expression level were significantly decreased (P<0.05). Compared with the model group, the weight of mice in VitD₃ low and high dose groups was increased significantly at each time point. The expression levels of H19, c-Myc mRNA, HMGA2 mRNA, the score of vimentin protein, and the expression levels of p-JAK2 and p-STAT3 protein in colon tissue were decreased significantly, while the let-7a expression and E-cadherin protein score were significantly increased (P<0.05). The TNF-α, IL-1β, IL-6 expression levels in serum were decreased, while the IL-10 expression level was increased (P<0.05). Changes were more obvious in VitD₃ high-dose group.Conclusion VitD₃ may play a protective role in CAC mice by regulating JAK2/STAT3 signal pathway, reducing inflammatory response and affecting EMT process.