In response to luteinizing hormone (LH), a higher concentration of progesterone (P4) is produced in luteal cells of corpus luteum (CL). Mitochondria are an essential cellular organelle in steroidogenesis. The specific engagement of the concept regarding mitochondrial shaping with early stages of steroidogenesis was suggested in reproductive endocrine cells. Although the specific involvement of GTPase dynamin-related protein 1 (Drp1) with steroidogenesis has been demonstrated in luteal cells of bovine CL in vitro, its actual relationship with ovarian steroidogenesis during the estrous cycle remains unknown. In this study, while Fis1 and Opa1 protein levels did not show significant changes during the estrous cycle, Drp1, Mfn1, and Mfn2 proteins exhibited relatively lower levels at proestrus than at estrus or diestrus. 3β-HSD showed higher levels at proestrus than at estrus or diestrus. In addition, Drp1 phosphorylation (s637) was higher in proestrus than in estrus or diestrus. Immune-positive cells for Drp1, pDrp1 (s637), and 3β-HSD were all localized in the cytoplasm of luteal cells in the CL. The immune-positive cells for 3β-HSD were more frequently seen in the CL at proestrus than at estrus or diestrus. Immunoreactivity for Drp1 in luteal cells at proestrus was weaker than that at estrus or diestrus. However, pDrp1 (s637) immune-positive cells were mostly detected in luteal cells at proestrus. These results imply that steroidogenesis (P4 production) in the CL is closely related to phosphorylation of Drp1 at serine 637. Taken together, this study presents evidence that Drp1 phosphorylation at serine 637 is an important step in steroidogenesis in the CL.