Update publication information. The ovary is the most important sexual organ of female mammals, and its corpus luteum plays a significant role in mammalian reproduction during the estrus cycle. Here we conducted a quantitative proteomic analysis using tandem mass tag (TMT)-labeling coupled with Nano-LC-MS/MS to explore the mechanisms of the estrus cycle in protein level. A total of 2,103 proteins were quantified in three groups, corpus hemorrhagicum (CH), corpus luteum (CL), and corpus fibrosum (CF). Thirty-two proteins were identified as differentially expressed in the CH group, and 116 proteins were identified as differentially expressed in the CF group, with the CL group serving as the control. Notably, we found that many enzymes, including kinase and phosphatase, are upregulated in the CL stage of the ovary, and three upregulated proteins in the CL stage (PLK1, PGP, and HGS) were confirmed using western blotting, quantitative RT-PCR, and immunohistochemistry analysis. The results of these validations were consistent with the TMT-label quantitative analysis, which indicated that they may play a crucial role in the normal reproductive cycle of the CL. Our study not only provides a deeper understanding of the formation and regression of the CL, but also suggests some potential proteins related to the estrus cycle.