Metformin, a cornerstone treatment for type 2 diabetes, has recently emerged as a multifunctional therapeutic agent with applications extending far beyond glycemic control. This mini-review explores its promising roles within extracellular matrix (ECM)-based three-dimensional (3D) cell culture models, which closely mimic the in vivo cellular microenvironment. Through modulation of key pathways—including AMPK activation, inhibition of TGF-β signaling, suppression of aerobic glycolysis, and regulation of microRNAs—metformin exerts profound effects on ECM remodeling, fibrosis attenuation, and tumor progression. Furthermore, its integration into ECM-inspired drug delivery systems and regenerative scaffolds has shown great potential in applications such as diabetic wound healing, bone repair, and implant integration. Collectively, current findings highlight metformin as a versatile agent capable of modulating ECM biology in disease-specific 3D systems, paving the way for innovative therapeutic strategies in oncology, metabolic disorders, and tissue engineering.