Conspectus[NiFe] hydrogenases catalyze reversible hydrogen production/consumption. The core unit of [NiFe] hydrogenase consists of a large and a small subunit. The active site of the large subunit of [NiFe] hydrogenases contains a NiFe(CN)2CO cluster. The biosynthesis/maturation of these hydrogenases is a complex and dynamic process catalyzed primarily by six Hyp proteins (HypABCDEF), which play central roles in the maturation process. HypA and HypB are involved in the Ni insertion, whereas HypC, D, E, and F are required for the biosynthesis, assembly, and insertion of the Fe(CN)2CO group. HypE and HypF catalyze the synthesis of the CN group through the carbamoylation and cyanation of the C-terminus cysteine of HypE. HypC and HypD form a scaffold for the assembly of the Fe(CN)2CO moiety.Over the last decades, a large number of biochemical studies on maturation proteins have been performed, revealing basic functions of each Hyp protein and the overall framework of the maturation pathway. However, it is only in the last 10 years that structural insight has been gained, and our group has made significant contributions to the structural biology of hydrogenase maturation proteins.Since our first publication, where crystal structures of three Hyp proteins have been determined, we have performed a series of structural studies of all six Hyp proteins from a hyperthermophilic archaeon Thermococcus kodakarensis, providing molecular details of each Hyp protein. We have also determined the crystal structures of transient complexes between Hyp proteins that are formed during the maturation process to sequentially incorporate the components of the NiFe(CN)2CO cluster to immature large subunits of [NiFe] hydrogenases. Such complexes, whose crystal structures are determined, include HypA-HypB, HypA-HyhL (hydrogenase large subunit), HypC-HypD, and HypC-HypD-HypE. The structures of the HypC-HypD, and HypCDE complexes reveal a sophisticated process of transient formation of the HypCDE complex, providing insight into the molecular basis of Fe atom cyanation. The high-resolution structures of the carbamoylated and cyanated forms of HypE reveal a structural basis for the biological conversion of primary amide to nitrile. The structure of the HypA-HypB complex elucidates nucleotide-dependent transient complex formation between these two proteins and the molecular basis of acquisition and release of labile Ni. Furthermore, our recent structure analysis of a complex between HypA and immature HyhL reveals that spatial rearrangement of both the N- and C-terminal tails of HyhL will occur upon the [NiFe] cluster insertion, which function as a key checkpoint for the maturation completion. This Account will focus on recent advances in structural studies of the Hyp proteins and on mechanistic insights into the [NiFe] hydrogenase maturation.