Pelvic organ prolapse (POP) is a condition that significantly affects women’s quality of life. The pathological mechanism of POP is not yet fully understood, and its pathogenesis is often caused by multiple factors, including the metabolic imbalance of the extracellular matrix (ECM). This study aims to investigate the role of miR-5195-3p, a microRNA, in the pathology of POP and its regulatory mechanism. Using various molecular biology techniques such as qRT-PCR, FISH, immunohistochemistry, and western blot, miR-5195-3p expression was examined in vaginal wall tissues obtained from POP patients. Results revealed an up-regulation of miR-5195-3p expression in these tissues, showing a negative correlation with the expression of ECM-related proteins. Further analysis using bioinformatics tools identified LOX as a potential target in POP. Dual luciferase reporter gene experiments confirmed LOX as a direct target of miR-5195-3p. Interestingly, regulating the expression of LOX also influenced the TGF-β1 signaling pathway and had an impact on ECM metabolism. This finding suggests that miR-5195-3p controls ECM metabolism by targeting LOX and modulating the TGF-β1 signaling pathway. In conclusion, this study unveils the involvement of miR-5195-3p in the pathological mechanism of POP by regulating ECM metabolism through the LOX/TGF-β1 axis. These findings reveal new mechanisms in the pathogenesis of pelvic POP, providing a theoretical foundation and therapeutic targets for further research on POP treatment.