Previously, we have demonstrated that major histocompatibility class II trans-activator (CIITA) is crucial in mediating interferon-gamma (IFN-gamma)-induced repression of collagen type I gene transcription. Here we report that CIITA represses collagen transcription through a phosphorylation-dependent interaction between its proline/serine/threonine domain and co-repressor molecules such as histone deacetylase (HDAC2) and Sin3B. Mutation of a serine (S373A) in CIITA, within a glycogen synthase kinase 3 (GSK3) consensus site, decreases repression of collagen transcription by blocking interaction with Sin3B. In vitro phosphorylation of CIITA by GSK3 relies on a casein kinase I site three amino acids C-terminal to the GSK3 site in CIITA. Both GSK3 and casein kinase I inhibitors alleviate collagen repression and disrupt IFN-gamma-mediated recruitment of Sin3B and HDAC2 to the collagen start site. Therefore, we have identified the region within CIITA responsible for mediating IFN-gamma-induced inhibition of collagen synthesis.