The actin cytoskeleton constitutes one of the most dynamic and versatile structural systems within eukaryotic cells, underpinning a broad range of essential biological processes including cell shape maintenance, migration, intracellular transport, signal transduction, and mechanical force generation. Far from being a static scaffold, actin filaments undergo continuous remodeling governed by a complex network of regulatory proteins, signaling cascades, and biomechanical cues. The precise spatial and temporal control of actin dynamics is fundamental to tissue development, homeostasis, and adaptive cellular responses. Disruption of this finely tuned regulatory system has increasingly been recognized as a central contributor to the pathogenesis of numerous human disorders, ranging from cancer and neurodegenerative diseases to cardiovascular, immunological, and developmental abnormalities. This review provides a comprehensive and integrative analysis of the molecular mechanisms governing actin cytoskeleton regulation and examines how their dysregulation drives disease-associated cellular phenotypes. Emphasis is placed on the interplay between actin-binding proteins, upstream signaling pathways, and mechanotransduction processes that collectively orchestrate cytoskeletal behavior. Furthermore, the emerging therapeutic potential of targeting actin-associated regulatory networks is critically discussed, highlighting both current limitations and future opportunities. By synthesizing insights from cell biology, molecular pathology, and translational research, this review underscores the actin cytoskeleton as a pivotal regulatory hub in human disease and a promising frontier for therapeutic intervention.