Simple SummaryThe gut microbiota can regulate lipid metabolism with its metabolic products through the gut–liver axis. In the present study, using an HFD-induced obese chicken model, we performed a multiple omics analysis using metabolomics and transcriptomics to identify gut–liver crosstalks involved in regulating the lipogenesis of chicken. The results showed that 5-hydroxyisourate, alpha-linolenic acid, bovinic acid, linoleic acid, and trans-2-octenoic acid might serve as signal molecules between the gut and liver. In the liver, they might enhance the expression ofACSS2,PCSK9, andCYP2C18and down-regulate one or more genes ofCDS1,ST8SIA6,LOC415787,MOGAT1,PLIN1,LOC423719, andEDN2to promote the lipogenesis of chicken. Moreover, taurocholic acid might be transported from the gut to the liver and contribute to HFD-induced lipogenesis by regulating the expression of ACACA, FASN, AACS, and LPL in the liver. This study lays the foundations for further elucidation of the gut–liver crosstalk mechanisms underlying lipogenesis in chickens.AbstractGrowing evidence has shown the involvement of the gut–liver axis in lipogenesis and fat deposition. However, how the gut crosstalk with the liver and the potential role of gut–liver crosstalk in the lipogenesis of chicken remains largely unknown. In this study, to identify gut–liver crosstalks involved in regulating the lipogenesis of chicken, we first established an HFD-induced obese chicken model. Using this model, we detected the changes in the metabolic profiles of the cecum and liver in response to the HFD-induced excessive lipogenesis using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) analysis. The changes in the gene expression profiles of the liver were examined by RNA sequencing. The potential gut–liver crosstalks were identified by the correlation analysis of key metabolites and genes. The results showed that a total of 113 and 73 differentially abundant metabolites (DAMs) between NFD and HFD groups were identified in the chicken cecum and liver, respectively. Eleven DAMs overlayed between the two comparisons, in which ten DAMs showed consistent abundance trends in the cecum and liver after HFD feeding, suggesting their potential as signaling molecules between the gut and liver. RNA sequencing identified 271 differentially expressed genes (DEGs) in the liver of chickens fed with NFD vs. HFD. Thirty-five DEGs were involved in the lipid metabolic process, which might be candidate genes regulating the lipogenesis of chicken. Correlation analysis indicated that 5-hydroxyisourate, alpha-linolenic acid, bovinic acid, linoleic acid, and trans-2-octenoic acid might be transported from gut to liver, and thereby up-regulate the expression ofACSS2,PCSK9, andCYP2C18and down-regulate one or more genes ofCDS1,ST8SIA6,LOC415787,MOGAT1,PLIN1,LOC423719, andEDN2in the liver to enhance the lipogenesis of chicken. Moreover, taurocholic acid might be transported from the gut to the liver and contribute to HFD-induced lipogenesis by regulating the expression ofACACA,FASN,AACS, andLPLin the liver. Our findings contribute to a better understanding of gut–liver crosstalks and their potential roles in regulating chicken lipogenesis.