The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant baculovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineal injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.