Corpus luteum (CL) regression is required for ovulation, with incomplete luteolysis associated with decreased fertility. Changes in gene expression have been correlated with CL from known days of the cycle and circulating progesterone (P4), and from exogenous prostaglandin F_2α (PGF) induced regression. However, characterization of gene expression in physiologic regression with knowledge of PGF exposure is lacking. To address this, daily CL biopsies were collected with bihourly blood samples for 74 h from d 18–21, and circulating P4 and PGFM (PGF metabolite) analyzed. Hormone profiles were used to classify biopsies in 3 groups: control: CL unexposed to PGF in the previous 24 h (C; n = 6); EL: CL with exposure to ≥1 PGFM pulse (n = 8); and LL: when P4 < 1 ng/mL (n = 5). Whole transcriptome RNA-seq raw data were aligned to bovine reference genome (NCBI, UMD3.1) with Tophat, followed by differential gene expression analysis using Cufflinks. Compared with control, 173 genes were differentially expressed (DE: Q < 0.05) in EL (103 upregulated, 70 downregulated), while 4615 were DE in LL (2455 upregulated, 2160 downregulated), with 161 genes DE in both EL and LL. Of these, 145 (84% DE genes in EL) displayed an increased fold change from control in LL compared with EL, showing most DE genes in early regression are further up or downregulated as regression progresses. For upregulated genes, enriched pathways in LL included hemostasis (P = 4.0E-8), extracellular matrix organization (P = 4.9E-8), and innate immune system (P = 2.1E-6), while pathways in downregulated genes were enriched in cholesterol biosynthesis (P = 7.1E-12), metabolism of lipids and lipoproteins (P = 1.0E-17), and steroid biosynthesis (P = 3.2E-6). While these results are largely confirmatory of pathways associated with luteolysis, this is the first study to characterize physiologic regression with serial biopsies and known PGF exposure. Furthermore, the small number of genes altered after small PGF pulses but before the drop in P4 associated with luteolysis suggests further inquiry into the role of small PGF pulses in physiologic luteolysis is warranted.