Objective This work is to construct a recombinant adeno-associated virus (rAAV) vector containing rat leptin gene, which mediates transduction of leptin into neurons in rat primary neuronal culture. Methods The leptin cDNA was obtained from rat adipose tissue by RT-PCR, cloned into pGEM-T vector, confirmed by sequencing, and then transferred into an AAV2 vector pTR-UF22 that carries CBA promotor. This vector, designated as pAAV2-CBA—Leptin, was packaged in human embryonic kidney (HEK) 293 cells using pDG as helper plasmid and purified with a single-step gravity-flow column. Copies of viral genome DNA were determined by quantitative PCR. Vector doses were expressed as viral genome copies (vg). The viral vectors AAV2-CBA-Leptin and AAV2-CBA-EGFP were transduced into primary cerebral cortical neuronal culture, and leptin expression determined using Western blotting. The cellular localization of EGFP in neuronal cultures was determined by immunocytochemistry. Results Restriction enzyme digestion and sequencing results demonstrate successful cloning of the amplified leptin cDNA into pGEM-T vector and subsequent subcloning into pTR-UF22. Incubation of cerebral cortical neuronal cultures with AAV2-CBA-leptin produced dose-dependent increases in leptin protein expression as compared to the background control with AAV2-CBA-EGFP. The AAV2-CBA-EGFP generated a high level of EGFP expression that colocalized with neuron-specific immunolabeling of NeuN, indicating a specificity of neuronal transduction by the vector. Conclusion We have developed a recombinant viral vector that efficiently transduces leptin into neurons. This vector offers a valuable bio-tool for studies of leptin CNS function in the control of body weight and diabetes.