Abstract: The study was carried out to investigate the effects of phospholipid and cholesterol on the hepatopancreas transcriptome of Procambarus clarkii. We designed nine diets containing three phospholipid（PL） le-vels（0, 20, 60 g/kg）and three cholesterol（CH） levels（0, 5, 10 g/kg） of diet, as a 3×3 experiment design, and marked as A～I. After 60 days of feeding, the hepatopancreas transcriptome analysis was conducted using RNA-Seq technology. 508 051 unigenes were selected and the average length was 521.6 bp. The differently expressed genes（DEGs）were screened with a threshold criterion of P ＜0.05. Several DEGs were randomly selected for validation using quantitative real-time PCR（RT-qPCR）. The sequence library of hepatopancreas samples from group I with high dosage（PL 60 g/kg; CH 10 g/kg）, group E with medium dosage（PL 20 g/kg; CH 5 g/kg） and group A with low dosage（PL 0 g/kg; CH 0 g/kg） were compared. The GO enrichment analysis showed that most of the differently expressed genes were mainly involved in growth, reproduction, immune system process, metabolic process and other pathways. KEGG pathway enrichment analysis suggested that significant enrichment in pathways including fat digestion and absorption, ovarian steroidogenesis, fatty acid biosynthesis, vitamin digestion and absorption, and glycerolipid metabolism. Differentially expressed genes linked to lipid metabolism and steroid hormone biosynthesis showed a significantly upregulation（P＜0.05）. This research provides reference data about the regulatory mechanism that the effects of dietary PL and CH on the growth and development of P.clarkii.