The vagina is a highly adaptable organ whose extracellular matrix (ECM) provides essential structural support throughout life, enabling functions such as intercourse, menstrual flow, and childbirth. This chapter describes the key ECM components in the vagina—collagens, elastic fibers, proteoglycans, glycoproteins, and enzymes—that together determine tissue function. Collagen types I and III form the main fibrillar framework and structural support, while elastic fibers enable tissue recoil and resilience needed during physiological events such as pregnancy, childbirth, and intercourse. Proteoglycans and glycosaminoglycans maintain tissue hydration and help the vagina resist compressive forces; also, matrix metalloproteinases and their inhibitors regulate ECM remodeling. The vaginal wall consists of four layers, including epithelium, subepithelium, muscularis, and adventitia, each contributing to structural integrity, flexibility, and function. ECM composition changes dynamically across developmental stages: menstruation, pregnancy, postpartum recovery, and menopause. Improper ECM remodeling correlates with pelvic organ prolapse and the genitourinary syndrome of menopause. Finally, the chapter discusses noninvasive imaging techniques for quantifying ECM composition, supporting the design of biomaterials that mimic native vaginal ECM and improve pelvic floor disorder outcomes.