Autophagy is an evolutionarily conserved pathway, in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation. Over 40 conserved autophagy-related (ATG) genes define the core machinery for the five processes of autophagy: initiation, nucleation, elongation, closure, and fusion. In this review, we focus on one of the least well-characterized events in autophagy, namely the closure of the isolation membrane/phagophore to form the sealed autophagosome. This process is tightly regulated by ESCRT machinery, ATG proteins, Rab GTPase and Rab-related proteins, SNAREs, sphingomyelin, and calcium. We summarize recent progress in the regulation of autophagosome closure and discuss the key questions remaining to be addressed.