The Phosphoinositide Specific-Phospholipase C (PI-PLC) family of enzymes plays a crucial role in various cellular processes by catalyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P] into inositol 1,4,5-trisphosphate (IP) and diacylglycerol (DAG), which are essential messengers mediating critical intracellular signaling pathways. Herein, we carry out a comprehensive analysis of the structure, function, regulation, and implications of the PI-PLC family enzymes in both physiological and pathological contexts. More specifically, we discuss the structural features of PI-PLCs, elucidating their conserved domains and catalytic mechanisms. Furthermore, we explore the multifaceted roles of PI-PLCs in signal transduction, cellular homeostasis, and membrane dynamics, whilst highlighting the intricate regulatory mechanisms governing their activity such as protein-protein interactions, post-translational modifications, and lipid modulation. Lastly, we assess the involvement of PI-PLCs in various diseases, such as cancer, neurological disorders, immune dysregulation, and male infertility, emphasizing their potential as therapeutic targets.