Glutamate dehydrogenases (GDHs) are widespread metabolic enzymes that play key roles in nitrogen homeostasis. Large glutamate dehydrogenases composed of 180kDa subunits (L-GDHs(180)) contain long N- and C-terminal segments flanking the catalytic core. Despite the relevance of L-GDHs(180) in bacterial physiology, the lack of structural data for these enzymes has limited the progress of functional studies. Here we show that the mycobacterial L-GDH(180) (mL-GDH(180)) adopts a quaternary structure that is radically different from that of related low molecular weight enzymes. Intersubunit contacts in mL-GDH(180) involve a C-terminal domain that we propose as a new fold and a flexible N-terminal segment comprising ACT-like and PAS-type domains that could act as metabolic sensors for allosteric regulation. These findings uncover unique aspects of the structure-function relationship in the subfamily of L-GDHs. Lazaro et. al. report the first 3D structure of a large glutamate dehydrogenase (L-GDH), the one corresponding to the Mycobacterium smegmatis enzyme composed of 180kDa subunits (mL-GDH(180)), obtained by X-ray crystallography and cryo-electron microscopy. This structure reveals that mL-GDH(180) assembles as tetramers with the N- and C-terminal domains being involved in inter-subunit contacts and unveils unique features of the subfamily of L-GDHs.