The study provides a comprehensive proteomic analysis of the human autophagy interaction network (AIN) under basal autophagy conditions, revealing 751 interactions among 409 candidate proteins. The AIN is characterized by extensive connectivity among sub-networks, including the UBL conjugation machinery and ATG8 sub-networks. The ATG8 family proteins, which are central to autophagosome assembly, interact with a cohort of 67 proteins, many of which are involved in vesicle trafficking, protein or lipid phosphorylation, and protein ubiquitination. The study also validates sub-networks within the AIN, such as the ULK1 protein kinase, PIK3C3/BECN1 kinase, and ATG2 sub-networks, and examines the role of ATG8 lipidation in sub-network assembly. RNAi analysis of the AIN shows that depletion of specific genes alters autophagosome formation, either by increasing or decreasing the number of autophagosomes. The findings provide a global view of the mammalian autophagy interaction landscape and offer insights into the mechanisms underlying this critical protein homeostasis pathway.The study provides a comprehensive proteomic analysis of the human autophagy interaction network (AIN) under basal autophagy conditions, revealing 751 interactions among 409 candidate proteins. The AIN is characterized by extensive connectivity among sub-networks, including the UBL conjugation machinery and ATG8 sub-networks. The ATG8 family proteins, which are central to autophagosome assembly, interact with a cohort of 67 proteins, many of which are involved in vesicle trafficking, protein or lipid phosphorylation, and protein ubiquitination. The study also validates sub-networks within the AIN, such as the ULK1 protein kinase, PIK3C3/BECN1 kinase, and ATG2 sub-networks, and examines the role of ATG8 lipidation in sub-network assembly. RNAi analysis of the AIN shows that depletion of specific genes alters autophagosome formation, either by increasing or decreasing the number of autophagosomes. The findings provide a global view of the mammalian autophagy interaction landscape and offer insights into the mechanisms underlying this critical protein homeostasis pathway.