Background: Diabetic nephropathy (DN) is the most common complication of diabetes mellitus (DM). The signal pathway and molecular mechanism of renal fibrosis are not fully understood. In the present study, we aimed to explore the function of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) in DN. Method: Mouse mesangial cells (MMCs) were treated with low glucose (LG) or high glucose (HG). TAK242 or short hairpin TLR4 (shTLR4) were employed to down-regulate Toll-like receptor 4 (TLR4). The effect of MFHAS1 knockdown or overexpression on fibrosis-related factors, inflammatory factors and TLR4 in MMCs were examined after transfecting with short hairpin RNA (shRNA) or MFHAS1 overexpressed plasmid, respectively. The expression levels of MFHAS1, inflammatory factors, fibrosis factors and TLR4 in db/db or streptozotocin (STZ) mice tissues and MMCs were examined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. The effect of MFHAS1 overexpression in vivo was also evaluated. Results: The expression of MFHAS1 in db/db or STZ mice and HG-treated MMCs were significantly increased compared with normal control mice and LG-treated MMCs. Overexpression of MFHAS1 inhibited the expression of inflammatory and fibrotic factors, while knockdown of MFHAS1 promoted them. MFHAS1 suppressed the activation of TLR4 pathway via inhibiting the expression of TLR4, and then alleviating inflammation and fibrosis in DN. MFHAS1 overexpression in vivo improved the symptoms of STZ-induced DN mice. Conclusion: The current study demonstrated that MFHAS1 relieved inflammation and renal fibrosis in DN mice via inhibiting TLR4. The results revealed that the MFHAS1 may be a molecular target in DN therapy.