AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis. Although AMPK has been studied extensively in cellular processes, understanding of its substrates and downstream functional network, and their contributions to cell fate and disease development, remains incomplete. To elucidate the AMPK-dependent signaling pathways, we performed global quantitative phosphoproteomic analysis using wild-type and AMPK1/2-double knockout cells and discovered 160 AMPK-dependent phosphorylation sites.