In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This depends on the ratio of pro- and anti-angiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23-K prolactin (PRL) in the bovine CL and its anti-angiogenic N-terminal fragments after extracellular cleavage by cathepsin D (Cath D). Prolactin RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells and steroidogenic cells. Cath D was detected in CL tissue, cell extracts and corresponding cell supernatants. In the intact CL, 23-K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early to late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF~FRLK(Dnp)-D-R-NH2 was cleaved by CL cell supernatants, providing further evidence for Cath D activity. 16-K PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion: (1) The bovine CL is able to produce PRL and to process it into anti-angiogenic fragments by Cath D activity. (2) PRL cleavage might mediate angioregression during luteolysis.