AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase that plays a crucial role in maintaining cellular energy homeostasis. Over the past decade, extensive research has identified various molecular mechanisms and physiological conditions that regulate AMPK activity. AMPK regulates diverse metabolic and physiological processes and is dysregulated in major chronic diseases such as obesity, inflammation, diabetes, and cancer. This review discusses the current understanding of the molecular and physiological regulation of AMPK and its metabolic and physiological functions. Additionally, it explores the mechanisms underlying AMPK's versatile roles in diabetes and cancer. AMPK is emerging as a promising target for preventing and treating these diseases due to its critical roles in physiology and pathology.AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase that plays a crucial role in maintaining cellular energy homeostasis. Over the past decade, extensive research has identified various molecular mechanisms and physiological conditions that regulate AMPK activity. AMPK regulates diverse metabolic and physiological processes and is dysregulated in major chronic diseases such as obesity, inflammation, diabetes, and cancer. This review discusses the current understanding of the molecular and physiological regulation of AMPK and its metabolic and physiological functions. Additionally, it explores the mechanisms underlying AMPK's versatile roles in diabetes and cancer. AMPK is emerging as a promising target for preventing and treating these diseases due to its critical roles in physiology and pathology.