Antigen presentation by human lymphocyte antigen (HLA) class II peptide receptors alerts the immune system to infections. In antigen‐presenting cells (APCs), HLA class II, HLA‐DM, and associated invariant chain‐encoding genes are exclusively regulated by the interferon γ (IFNγ)‐inducible class II transactivator (CIITA). Control of CIITA expression could therefore govern expression of class II peptide receptors in the diverse group of APCs. We discovered that elevation of the HLA class III region encoded B‐associated transcript 3 (BAT3) increases and depletion of BAT3 decreases expression of HLA class II, HLA‐DM, and invariant chain. IFNγ strongly elevates BAT3 transcription in various tumor cell lines and in primary macrophages. BAT3 chaperones the simultaneously IFNγ‐induced CIITA. Following IFNγ‐treatment, both CIITA and BAT3 translocate from the cytosol to the nucleus. The nuclear import of CIITA mediated by IFNγ controls activation of HLA class II genes. BAT3 is a novel key regulator of components of the HLA class II processing pathway. We present a mechanism explaining how parallel IFNγ‐mediated regulation of CIITA and of its chaperone BAT3 controls the level of components of the HLA class II processing pathway.—Kämper, N., Franken, S., Temme, S., Koch, S., Bieber, T., Koch, K. γ‐Interferon‐regulated chaperone governs human lymphocyte antigen class II expression. FASEB J. 26, 104–116 (2012). www.fasebj.org