Background: During cholestatic liver diseases, cholangiocytes express neuroendocrine phenotypes, and respond to a number of hormones/neuropeptides by paracrine and autocrine mechanisms. We examined whether the neuroendocrine hormone progesterone is produced by and targeted to cholangiocytes thereby regulating biliary proliferation during cholestasis. Methods: Nuclear (PR-A and PR-B) and membrane (PRGMC1, PRGMC2 and mPR) progesterone receptor expression was evaluated in liver sections and cholangiocytes from normal and BDL rats, and NRC cells (normal rat cholangiocyte line). In vivo, normal rats were chronically treated with progesterone for 1 week, or immediately after BDL, rats were treated with a neutralizing progesterone antibody for 1 week. Cholangiocyte growth was measured by evaluating the number of bile ducts in liver sections. The expression of the progesterone synthesis pathway was evaluated in liver sections, cholangiocytes and NRC. Progesterone secretion was evaluated in supernatants from normal and BDL cholangiocytes and NRC. In vitro, NRC were stimulated with progesterone, and cholangiocyte supernatants in the presence or absence of anti-progesterone antibody. Aminoglutethimide was used to block progesterone synthesis. Results: Cholangiocytes and NRC express the PR-B nuclear receptor and PRGMC1, PRGMC2 and mPR. In vivo, progesterone increased the number of bile ducts of normal rats, whereas anti-progesterone antibody inhibited cholangiocyte growth stimulated by BDL. Normal and BDL cholangiocytes expressed the biosynthetic pathway for and secrete progesterone. In vitro: (i) progesterone increased NRC proliferation; (ii) cholangiocyte supernatants increased NRC proliferation, which was partially inhibited by preincubation with anti-progesterone; and (iii) inhibition of progesterone steroidogenesis prevented NRC proliferation. Conclusions: Progesterone may be an important autocrine/paracrine regulator of cholangiocyte proliferation.