Abstract: In order to screen and verify the key genes that isomaltulose reduces liver fat accumulation in mice, in this study, the Limma package was used to analyze the liver transcriptome data of male mice fed with isomaltose and sucrose for 22 weeks (GSE54723, n=14). A total of 49 DEGs were screened out, and then were analyzed by Metascape software for GO functional enrichment analysis and KEGG signaling pathways analysis. Cytoscape and WGCNA software were used to construct gene co-expression network, overlapping genes were picked up for searching for key node genes, and 10 key node genes Pnliprp1, Prss2, Clps, Tff2, Pnlip, Ctrl, Cpa1, Cel, Dmbt1, Vil1 were screened out, gene functional analysis showed Pnlip, Pnliprp1, Cel, Clps were related to lipid metabolism. The qPCR results showed that the expression of these four genes related to lipid metabolism in liver tissue of isomaltulose group was significantly (P<0.05) higher than that of sucrose group, by comparing the expression of the above genes in the liver transcriptome data of normal people and NAFLD patients (GSE163211, n=318), Pnlip, Pnliprp1, Cel and Clps were also expressed significantly higher in normal people than in NAFLD patients. This study revealed that isomaltulose promoted lipolysis by up regulating the synthesis of Pnlip, Pnliprp1, Cel and Clps, so as to reduce the accumulation of liver lipids, which would provide a theoretical support for the study of the molecular mechanism of isomaltulose affecting liver lipid metabolism.