Abstract Background T cells play a key role in the pathogenesis of chronic inflammatory enteropathy (CIE) in dogs. Cluster of differentiation 3 (CD3) antigen serves as a marker for T cells. In human medicine, Ki-67 is an indicator for cell growth but there are only a few studies in dogs with CIE. Objective To investigate Ki-67 in relation to T cells as a marker for CIE in dogs. Animals Eleven dogs with CIE and 6 healthy beagle controls (CO). Methods Retrospective case-control study. Dogs were clinically assessed by the Canine Chronic Enteropathy Clinical Activity Index (CCECAI). Duodenal mucosal biopsy samples were endoscopically obtained for histopathologic examination by means of the World Small Animal Veterinary Association score. Double-labeled immunofluorescence was used to investigate colocalization of Ki-67 and CD3 in epithelium and lamina propria (LP) of villi and crypts. Results Dogs with CIE had significantly higher clinical score (median, 5.0; interquartile range [IQR], 3-7) compared to CO (all 0; P < .001). The Ki-67/CD3 double-positive cells were significantly increased in the LP of the crypt region of CIE dogs (0.63 cells/mm2; IQR, 0-0.54) versus CO (0.08 cells/mm2; IQR, 0-0.26; P = .044). A significant correlation was found between CCECAI and the Ki-67/CD3 ratio in the LP of the crypt region (r = 0.670; P = .012) in dogs with CIE. Conclusions and Clinical Importance The Ki-67/CD3 ratio is upregulated in the LP crypt region of dogs with CIE and it correlates with clinical severity. Therefore, Ki-67/CD3 could be a useful tool for detection of CIE.