<p>The deposition of ectopic lipids and resulting insulin resistance (IR) are strongly linked to a high risk of many chronic metabolic diseases, such as type 2 diabetes mellitus (T2D) and metabolic dysfunction–associated steatotic liver disease (MASLD). Perilipin family proteins (PLINs), which flexibly regulate the storage and release of lipids contained in lipid droplets in tissues, as well as the connection between lipid droplets and mitochondria, enabling fatty acids to enter mitochondria for β-oxidation. This review provides an updated consolidation of the evolving role of PLINs in lipid metabolism. Exercise, effective in activating lipolysis in tissues, alleviates the deposition of ectopic lipids and IR. Exercise-induced metabolic adaptations can, in turn, modulate PLIN expression in multiple tissues. However, there are still some controversies regarding the changes in PLINs in different tissues during exercise and their role in regulating lipid metabolism. We comprehensively discuss the roles of PLINs in metabolically active tissues (the adipose tissue, liver, and skeletal muscle), the effect of exercise on PLIN expression, and the role of PLIN in reducing ectopic fat deposition during exercise. The interplay between PLIN expression, lipid dynamics, and metabolic states such as exercise, fasting, and obesity is consolidated to ascertain the importance of PLINs in regulating lipid metabolism. By synthesizing fragmented insights on PLINs into a coherent narrative, we attempted to connect PLIN function with exercise physiology and metabolic diseases and suggest potential avenues for research, diagnosis, and therapy.</p>