The objective of the present study was to investigate whether tissue inhibitor of metalloproteinase-3 (TIMP-3), pituitary adenylate cyclase-activating polypeptide (PACAP), and vasoactive intestinal peptide (VIP) participate in the occurrence of female stress urinary incontinence (SUI) by measuring the expression levels of TIMP-3, PACAP, and VIP in the vaginal wall and analyzing their correlation to understand the pathogenesis of female SUI. Forty female patients who were admitted to our hospital for tension-free obturator tape surgery for treatment of SUI from April, 2012 to December, 2015 were selected as the study group. Forty patients who underwent vaginal or total abdominal hysterectomy for treatment of non-estrogen-related diseases during the same period were selected as the control group. Tissue samples from the anterior vaginal wall, located at twelve o'clock, were taken from both groups. The expression levels of TIMP-3, PACAP and VIP were detected by immunohistochemistry, and the correlation of integral optical density (IOD) among expressions of TIMP-3, PACAP, and VIP was investigated. The expression of TIMP-3 in vaginal wall tissues of the study group was lower than that of the control group (P<0.05). The expression of PACAP and VIP in vaginal tissues of the study group were lower than those of the control group (P<0.05). In the study group, the IOD of PACAP expression was significantly and positively correlated with that of VIP (r=0.873, P<0.05), the IOD of PACAP expression was significantly and positively correlated with that of TIMP-3 (r=0.802, P<0.05), and the IOD of VIP expression was significantly and positively correlated with that of TIMP-3 (r=0.716, P<0.05). In conclusion, TIMP-3, PACAP and VIP jointly participate in the occurrence of female SUI. Increasing the expression of TIMP-3, PACAP, and VIP, repairing neurons, and enhancing the elasticity of vaginal wall tissues may become a new way to treat female SUI.