Bovine endometritis can lead to abnormal endometrial function and fibrosis, resulting in difficulties in successful embryo implantation and intrauterine adhesions. Melatonin is well known for its profitable effects against inflammation and pathological fibrosis in discrepant organs. Considering the potential therapeutic benefits of melatonin, this study aimed to investigate its effects on endometrial fibrosis in cows with endometritis. Firstly, we evaluated the expression patterns of various factors associated with fibrosis, such as transforming growth factor-β1 (TGF-β1), extracellular matrix (ECM)-related markers (COL1A1 and COL3A1), epithelial-mesenchymal transformation (EMT)-related proteins (α-SMA and Vimentin) in healthy and endometritis-affected bovine uterine tissues. The results showed that diseased tissues presented significantly higher TGF-β1 expression, ECM production, and EMT progression versus normal tissues. Moreover, we established an LPS-induced fibrosis model in endometrial stromal cells (ESCs), and found that melatonin inhibited the fibrosis process of ESCs in a dose-dependent manner. The MT2 inhibitor 4P-PDOT blocked the antifibrotic effects of melatonin and inhibited the phosphorylation of Smad2/3, ERK1/2, and JNK1/2 in LPS-induced fibrosis of ESCs, but not P38 MAPK. These data implied that melatonin supplementation attenuated LPS-induced fibrosis in ESCs by modulating the inhibition of TGF-β/Smad, ERK, and JNK signaling pathways via MT2.