Abstract: The purpose of this study was to analyze the gene expression profiles in the hypothalamus of beef cattle related to high and low residual feed intake（residual feed intake, RFI）, and to explore the relationship between the difference of gene expression and the phenotypic variation of bovine RFI. Three HRFI and three LRFI Qinchuan cattle were selected and divided into high residual feed intake（HRFI） group and low residual feed intake（LRFI） group. Hypothalamic tissue samples were collected and total RNA was extracted for transcriptome sequencing. RFI-related differential genes were screened and analyzed by GO functional annotation and KEGG pathway enrichment analysis. The protein interaction network map of differentially expressed genes was constructed by using STRING database. Finally, six differential genes were randomly selected for real-time fluorescence quantitative verification. The results showed that there were 1 538 differentially expressed genes in HRFI group and LRFI group, including feeding-related genes such as AGRP and PMCH, and metabolism-related genes such as GH1, MTNR1A and RYR2. In addition, the differential genes were mainly enriched in calcium signal pathway, cAMP signal pathway and circadian rhythm. Three core subnetworks and seven core genes were screened by protein interaction network. RT-qPCR verification showed that the quantitative results were consistent with the sequencing results. It is indicated that the differential genes such as AGRP, PMCH, GH1, MTNR1A and RYR2 in hypothalamus and calcium signal pathway, cAMP signal pathway and circadian rhythm might be involved in the regulation of RFI in beef cattle.