Chaperone-mediated autophagy (CMA) is a selective proteolytic pathway in lysosomes that plays a crucial role in maintaining cellular homeostasis and regulating specific functions in various tissues. CMA substrates are recognized by the molecular chaperone Hsc70 and delivered to the lysosomal receptor LAMP-2A, which is the limiting component of this pathway. Post-translational modifications can generate or complete the KFERQ-like motif, increasing the number of potential CMA substrates. CMA is regulated by signaling pathways that modulate LAMP-2A levels and activity, such as the mTORC2-AKT1-PHLP1 axis. CMA has essential roles in protein quality control, cellular energetics, cellular reprogramming, and defense mechanisms. Its dysfunction is implicated in neurodegenerative diseases, cancer, and aging-associated diseases. In neurodegenerative diseases, CMA failure leads to protein aggregation and toxicity, contributing to disease progression. In cancer, CMA activity can be both pro-tumor and anti-tumor, depending on the context. Targeting CMA has emerged as a potential therapeutic strategy for various pathologies, but further research is needed to understand its complex regulatory roles and to develop selective modulators.Chaperone-mediated autophagy (CMA) is a selective proteolytic pathway in lysosomes that plays a crucial role in maintaining cellular homeostasis and regulating specific functions in various tissues. CMA substrates are recognized by the molecular chaperone Hsc70 and delivered to the lysosomal receptor LAMP-2A, which is the limiting component of this pathway. Post-translational modifications can generate or complete the KFERQ-like motif, increasing the number of potential CMA substrates. CMA is regulated by signaling pathways that modulate LAMP-2A levels and activity, such as the mTORC2-AKT1-PHLP1 axis. CMA has essential roles in protein quality control, cellular energetics, cellular reprogramming, and defense mechanisms. Its dysfunction is implicated in neurodegenerative diseases, cancer, and aging-associated diseases. In neurodegenerative diseases, CMA failure leads to protein aggregation and toxicity, contributing to disease progression. In cancer, CMA activity can be both pro-tumor and anti-tumor, depending on the context. Targeting CMA has emerged as a potential therapeutic strategy for various pathologies, but further research is needed to understand its complex regulatory roles and to develop selective modulators.