Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition. Two crystal structures of ALDH9A1 are reported, including the first of the enzyme complexed with NAD. One of the structures reveals the active conformation of the enzyme, in which the Rossmann dinucleotide-binding domain is fully ordered and the inter-domain linker adopts the canonical β-hairpin observed in other ALDH structures. The oligomeric structure of ALDH9A1 was investigated using analytical ultracentrifugation, small-angle X-ray scattering, and negative stain electron microscopy. These data show that ALDH9A1 forms the classic ALDH superfamily dimer-of-dimers tetramer in solution. Our results suggest that the presence of an aldehyde substrate and NAD promotes isomerization of the enzyme into the active conformation.