Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is an E3 ligase of ubiquitin fold modifier 1 (UFM1), which can act together with its target protein to inhibit the apoptosis of cells. Lipopolysaccharides (LPS) can affect the ovarian health of female animals by affecting the apoptosis of ovarian granulosa cells. The physiological function of UFL1 on the apoptosis of bovine (ovarian) granulosa cells (bGCs) remains unclear; therefore, we focused on the modulating effect of UFL1 on the regulation of LPS-induced apoptosis in ovarian granulosa cells. Our study found that UFL1 was expressed in both the nucleus and cytoplasm of bGCs. The results here demonstrated that LPS caused a significant increase in the apoptosis level of bGCs in cows, and also dramatically increased the expression of UFL1. Furthermore, we found that UFL1 depletion caused a significant increase in apoptosis (increased the expression of BAX/BCL-2 and the activity of caspase-3). Conversely, the overexpression of UFL1 relieved the LPS-induced apoptosis. In order to assess whether the inhibition of bGCs apoptosis involved in the nuclear factor-κB (NF-κB) signaling pathway resulted from UFL1, we detected the expression of NF-κB p-p65. LPS treatment resulted in a significant upregulation in the protein concentration of NF-κB p-p65, and knockdown of UFL1 further increased the phosphorylation of NF-κB p65, while UFL1 overexpression significantly inhibited the expression of NF-κB p-p65. Collectively, UFL1 could suppress LPS-induced apoptosis in cow ovarian granulosa cells, likely via the NF-κB pathway. These results identify a novel role of UFL1 in the modulation of bGC apoptosis, which may be a potential signaling target to improve the reproductive health of dairy cows.