ABSTRACT Under conditions of low oxygen availability, Neisseria meningitidis and Neisseria gonorrhoeae are able to respire via a partial denitrification pathway in which nitrite is converted to nitrons oxide. In this process, nitrite reductase (AniA), a copper (Cu)‐containing protein converts nitrite to NO, and this product is converted to nitrous oxide by nitric oxide reductase (NorB). NorB also confers protection against toxic NO, and so we devised a conditional lethal screen, using a norB mutant, to identify mutants that were resistant to nitrite‐dependent killing. After random‐deletion mutagenesis of N. meningitidis, this genetic screen identified a gene encoding a Cu chaperone that is essential for AniA function, AccA. Purified AccA binds one Cu (I) ion and also possesses a second binding site for Cu (II). This novel periplasmic Cu chaperone (AccA) appears to be essential for provision of Cu ions to AniA of pathogenic Neisseria to generate an active nitrite reductase. Apart from the Neisseria genus, AccA is distributed across a wide range of environmental Proteo‐bacteria species.—Jen, F. E.‐C., Djoko, K. Y., Bent, S. J., Day, C. J., McEwan, A. G., Jennings, M. P. A genetic screen reveals a periplasmic copper chaperone required for nitrite reductase activity in pathogenic Neisseria. FASEB J. 29, 3828‐3838 (2015). www.fasebj.org