Objective To investigate the effects of nicotine on the gene expression profile in prefrontal cortex (PFC) region of rats. Methods Twenty-four adult male F344 rats were randomly divided into treatment group and control group, with 12 animals in each group. Animals in treatment group were injected with nicotine solution while those in control group were given physiological saline for 17 days. At the end of drug administration, the tissue of PFC region was sliced from the brain of each animal. RNA sequencing (RNA-Seq) was utilized to analyze the gene expression profile in PFC of each animal. Bioinformatics tools including Bowtie, Tophat and Cufflinks were used to map the sequencing reads to the reference genome of rat, and to measure the relative expression level of each transcript. Genes whose expression levels were significantly differ-ent between the two groups were identified. The functional categories and the biochemical pathways associated with these genes were further analyzed. Results By comparing the expression level of each transcript between two groups, 885 signifi-cantly differentially expressed genes were identified. These genes were enriched in multiple Gene Ontology Biological Pro-cess categories (e.g., G protein coupled receptor protein signaling pathway, protein ubiquitination, and neurotransmitter up-take) and biological pathways (e.g., insulin signaling pathway, Alzheimer's disease, and long term potentiation). Conclu-sion Nicotine treatment may regulate signaling transduction, energy metabolism and other biological processes in PFC of rat brain.