In recent studies, UFL1 (ubiquitin-like modifier 1 ligating enzyme 1) has been identified as a significant regulator of NF-κB signaling and cellular stress response, yet its physiological function in LPS-stimulated bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the modulating effect of UFL1 on the regulation of LPS-induced inflammation and cell damage, with a focus on apoptosis, ER stress, autophagy, oxidative stress, and the TLR4/NF-κB signaling pathway. The results showed that UFL1 depletion aggravated the LPS-induced inflammatory response and cell damage by positively regulating the TLR4/NF-κB pathway (increased the expression of TLR4, NF-κB P65 in nuclear, and phospho-IκBα), exacerbating LPS-induced ER stress (increased the expression of CHOP, Hsp70, and GRP78), apoptosis (increased the expression of Bax/Bcl-2 and activity of caspase-3), autophagy (increased LC3-II and decreased P62 expression), and oxidative stress (decreased SOD and CAT levels and increased MDA levels). Overexpression of UFL1 suppressed the activation of the TLR4/NF-κB pathway and relieved the LPS-induced ER stress, apoptosis, autophagy, and oxidative stress, thereby alleviating the inflammatory response and cell damage. Collectively, UFL1 may play an important role during the inflammatory response and thereby acts as a potential therapeutic target for bovine mastitis.