Exogenous delivery of nerve growth factor (NGF) promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-beta (hNGF-beta) DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-beta adenoviral vector containing full-length hNGF-beta cDNA was generated by homologous recombination in Escherichia Coli. The rhNGF-beta adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-beta adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-beta adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-beta was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-beta adenovirus-infected cells (primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts) compared with non-infected cells. Total mRNA isolated from rhNGF-beta adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-beta adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-beta adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.