The study investigated the effects of adipose tissue-derived stem cells (ADSCs) modified with penile neuronal nitric oxide synthase (PnNOS) gene on intracellular calcium concentration in rat corpus cavernosum smooth muscle cells (CCSMCs). ADSCs and CCSMCs of Sprague-Dawley (SD) rats were isolated and cultured in vitro respectively. The rat PnNOS gene was transferred into the ADSCs mediated by a recombinant adenovirus vector. The expression of the PnNOS gene was detected. At the same time, the concentration of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was assayed. After coculturing with the CCSMCs of SD rats, which were isolated and expanded ex vivo, the cGMP and NO levels of ADSCs and CCSMCs were measured. Intracellular calcium concentration ([Ca2+](i)) in rat CCSMCs was measured with Fluo-3/AM by flow cytometer after cocultured with ADSCs overexpressing PnNOS gene. The mRNA and protein expression of PnNOS gene mediated by recombinant adenovirus vector significantly overexpressed and lasted at least 2weeks. Meanwhile, the concentration of NO and cGMP in ADSCs was greatly increased. The concentration of cGMP was significantly increased, and [Ca2+](i) was obviously decreased in CCSMCs compared with the control groups (P<0.05) after cocultured with ADSCs for 3days. These findings demonstrated that ADSCs overexpressing PnNOS gene might decrease [Ca2+]i in CCSMCs by up-regulating NO-cGMP signalling pathway.