Abnormal intestinal mucosal immunity plays a crucial role in ulcerative colitis (UC). Autotaxin (ATX) can promote T cell migration and was reported to have a regulatory effect on Th17 cells, while sphingosine-1-phosphate (S1P) and its receptors (S1PRs) modulate Th17/Treg balance and inflammation, with S1PR modulators approved for UC. ATX can catalyze sphingosylphosphorylcholine (SPC) to produce S1P; however, the relationship between ATX and S1P/S1PRs in UC is unclear. Understanding the role of ATX-S1P/S1PRs in intestinal immunity can provide new treatment strategies for intestinal inflammatory diseases. Both UC patients and DSS-induced colitic mice showed significantly increased levels of ATX and S1P compared with healthy controls. ATX inhibitor PF8380 treatment led to reduced levels of S1P/S1PRs in colitic mice. Consistent with this, the S1PR antagonist etrasimod was able to alleviate ATX-induced intestinal inflammation, as well as partially restore ATX-induced Th17/Treg imbalance in MLNs and the spleen. In HT-29 and Raw246.7 cells, ATX treatment led to enhanced expression of S1P/S1PRs, with S1PR1 being the most significant. Furthermore, S1PR1 mediates the effect of ATX on Th17/Treg cell differentiation and function in vivo. Therefore, ATX affects the differentiation and function of Th17/Treg cells through S1P/S1PR1 signaling, increased ATX expression leading to Th17/Treg cell imbalance, intestinal mucosal immune dysfunction, and exacerbating intestinal inflammation.