NKT cells are activated by CD1d and show an immune regulating function. Here, we investigated whether DX5(+)NKT cells could be used to reduce colitis in a chronic colitis mouse model and studied the potential immunological mechanisms involved. Chronic colitis was induced either by transfer of enriched CD62L(+)CD4(+) T cells to severe-combined-immunodeficient mice or by feeding dextran sodium sulfate to immune competent mice. DX5+NKT cells were transferred to mice with chronic colitis. Co-transfer of DX5(+)NKT cells, but not CD8(+) control cells, prevented the onset of colitis, and the immune regulatory effect of DX5(+)NKT cells was completely abrogated by injecting CD1d blocking antibody. Moreover, DX5(+)NKT cells reduced established colitis in both chronic colitis models. In vitro, DX5(+)NKT cells induced cell death of colon-infiltrating lymphocytes isolated from diseased mice. This effect was inhibited in the presence of either anti-CD1d or anti-programmed death ligand-1 (PD-L1) blocking antibodies. The specific potency of DX5(+)NKT cells in regulating chronic colitis in two mouse models is demonstrated. In vitro testing suggests that DX5(+)NKT cells activated by CD1d induce cell death of colitis-inducing lymphocytes, which is mediated through PD-L1. Therefore, DX5(+)NKT cells could be important in the regulation of immune responses associated with chronic colitis.