The corpus luteum (CL) is an exquisitely regulated transitory endocrine gland necessary for the onset and maintenance of pregnancy in mammals. Most of the data on the mechanisms of CL differentiation at the molecular level come from genomic studies, but direct protein data are scarce. Here we have undertaken a differential expression proteomic approach to identify, in an unbiased way, those proteins whose levels change significantly in the rat CL as it evolves from functionality during pregnancy to regression after parturition. Moreover, we have compared the regressing CL with the newly formed functional CL that coexist during lactation under the same endocrine environment. We have defined a “proteomic signature” of CL functionality, which is constituted by a set of 24 proteins with a few differences between pregnancy and lactation. Most of these markers are new and are involved in microtubule assembly, retinoic acid transport, and Raf kinase signaling cascade; 10 are enzymes that define a ketogenic metabolic landscape, demonstrating, for the first time, the prevalence of de novo cholesterol synthesis in luteal cells. The “proteomic signature of regression,” on the other hand, is composed of nine proteins, one of which is 20alpha-hydroxysteroid dehydrogenase and two, ferritin and gamma-actin, are new. The discovery of unpredictable new actors in the differentiation process of CL reported here will contribute to new hypotheses that explain the complex female reproductive function at the protein level. It will also open new doors to research on each identified protein by relating them to cellular differentiation.