Introduction.We have confirmed the higher expression of insulin-like growth factor binding protein-3 (IGFBP-3) in corpus cavernosal tissues of old rat and it may implicate for erectile dysfunction (ED).We further determined the effects of knockout of IGFBP-3 on the proliferation, the intracellular calcium concentration and the secretion of insulin-like growth factor-1 (IGF-1) in cavernosal smooth muscle cells (CSMCs) in vitro.Aim.To determine whether knockout of IGFBP-3 with plasmid-based short hairpin RNA (shRNA) can promote proliferation, affect the level of intracellular calcium concentration,guanosine 3',5'-cyclic-monophosphate (cGMP) and the secretion of IGF-1 in CSMCs.Main Outcome Measures.The cell proliferation, the fluorescence intensity (FI) of measurement of intracellular calcium, the cGMP formation and the secretion of IGF-1 were determined.Methods.Four different IGFBP-3 specific shRNAs (526,611, 710 and 866) and the negative control (NC) sequence were generated and four IGFBP-3-shRNAs and NC-shRNA were constructed.The validation of plasmid-based shRNA to knockout of IGFBP-3 were confirmed by western blot and quantitative real-time polymerase chain reaction (PCR) assays in CSMCs.The cell proliferation was measured by Methylthiazoletetrazolium (MTT) assay.The effect of shRNA on the level of intracellular calcium concentration, cGMP and the secretion of IGF-1 were assessed.Results.Western blot and quantitative PCR demonstrated that endogenous IGFBP-3 expression was efficiently suppressed with the three of four different IGFBP-3-shRNAs.Specific shRNA against IGFBP-3 significantly promoted the cell proliferation in CSMCs in time-dependent manner.Furthermore, the plasmid-based shRNA successfully decreased levels of intracellular calcium concentration in CSMCs.However, the IGFBP-3-shRNA increases the formation of the cGMP and the secretion of the IGF-1 in CSMCs.Conclusions: We conclude that the plasmid-based shRNA against IGFBP-3 can promote the CSMCs proliferation, attenuate the intracellular calcium concentration and increase the cGMP formation and IGF-1 secretion.It might play a role in regulating erectile function.