Background: We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection. Materials and Methods: Wildtype (WT) and DPIV-/- mice consumed 2% DSS in drinking water for 6 days to induce colitis. Mice were treated with saline or the DP inhibitors Ile-Pyrr-(2-CN)*TFA or Ile-Thia. DP mRNA and enzyme levels were measured in the colon. Glucagon-like peptide (GLP)-2 and GLP-1 concentrations were determined by radioimmunoassay, regulatory T-cells (Tregs) by fluorescence activated cell sorting (FACS) on FOXp3+T cells in blood, and neutrophil infiltration assessed by myeloperoxidase (MPO) assay. Results: DP8 and DP2 mRNA levels were increased (P < 0.05) in WT+saline mice compared to untreated WT mice with colitis. Cytoplasmic DP enzyme activity was increased (P < 0.05) in DPIV-/- mice at day 6 of DSS, while DP2 activity was increased (P < 0.05) in WT mice with colitis. GLP-1 (63%) and GLP-2 (50%) concentrations increased in WT+Ile-Pyrr-(2-CN)*TFA mice compared to day-0 controls. MPO activity was lower in WT+Ile-Thia and WT+Ile-Pyrr-(2-CN)*TFA treated mice compared to WT+saline (P < 0.001) at day 6 colitis. Conclusions: DP expression and activity are differentially regulated during DSS colitis, suggesting a pathophysiological role for these enzymes in human inflammatory bowel disease (IBD). DP inhibitors impaired neutrophil recruitment and maintenance of the Treg population during DSS-colitis, providing further preclinical evidence for the potential therapeutic use of these inhibitors in IBD. Finally, DPIV appears to play a critical role in mediating the protective effect of DP inhibitors.