Post-translational modifications of histones such as methylation, acetylation, and phoshorylation regulate chromatin structure and gene expression. However, phosphorylation of histone H3 at threonine 11 (H3T11ph) has not been linked to transcriptional regulation. Here we show that protein kinase C-related kinase 1 (PRK1) phosphorylates H3T11 upon ligand-dependent recruitment to androgen receptor (AR) target genes. PRK1 is pivotal to AR function since PRK1 knockdown by RNAi or PRK1 inhibition by treatment with Ro318220 impedes AR-dependent gene expression. Blocking PRK1 function abrogates androgen-induced H3T11 phosphorylation, and in consequence, inhibits androgen-induced demethylation of histone H3 in vivo. Moreover, the presence of serine 5-phosphorylated RNA polymerase II is no longer observed at AR target promoters. In vitro, phosphorylation of H3T11 by PRK1 accelerates demethylation by the Jumonji C (JmjC) domain-containing protein JMJD2C3. Thus, phosphorylation of H3T11 by PRK1 establishes a novel chromatin mark for transcriptional activation, identifying PRK1 as a gatekeeper of AR-regulated gene expression. Importantly, elevated PRK1- and H3T11ph levels positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PRK1 blocks AR-induced tumour cell proliferation. Thus, PRK1 is a promising therapeutic target in the treatment of prostate cancer.