BackgroundGut microbiota are involved in the onset and development of chronic intestinal inflammation. The recently described endocannabinoidome (eCBome), a diverse and complex system of bioactive lipid mediators, has been reported to play a role in various physio-pathological processes such as inflammation, immune responses and energy metabolism. The eCBome and the gut microbiome (miBIome) are closely linked and form the eCBome - miBIome axis, which may be of special relevance to colitis.MethodsColitis was induced in conventionally raised (CR), antibiotic-treated (ABX) and germ-free (GF) mice with dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by Disease Activity Index (DAI) score, body weight change, colon weight-length ratio, myeloperoxidase (MPO) activity and cytokine gene expression. Colonic eCBome lipid mediator concentrations were measured by HPLC-MS /MS.ResultsGF mice showed increased levels of anti-inflammatory eCBome lipids (LEA, OEA, DHEA and 13- HODE-EA) in the healthy state and higher MPO activity. DNBS elicited reduced inflammation in GF mice, having lower colon weight/length ratios and lower expression levels ofIl1b,Il6,Tnfaand neutrophil markers compared to one or both of the other DNBS-treated groups.Il10expression was also lower and the levels of severalN-acyl ethanolamines and 13-HODE-EA levels were higher in DNBS-treated GF mice than in CR and ABX mice. The levels of these eCBome lipids negatively correlated with measures of colitis and inflammation.ConclusionsThese results suggest that the depletion of the gut microbiota and subsequent differential development of the gut immune system in GF mice is followed by a compensatory effect on eCBome lipid mediators, which may explain, in part, the observed lower susceptibility of GF mice to develop DNBS-induced colitis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12944-023-01823-1.