Renal fibrosis, which is characterized by excessive extracellular matrix (ECM) accumulation in the renal tubulointerstitium, can lead to chronic kidney disease (CKD). The role of microfiber-associated protein 4 (MFAP4), which is an ECM protein that interacts with elastin and collagen, in renal fibrosis has not been investigated. The aim of this study was to examine the role of MFAP4 in the pathogenesis of renal fibrosis and the underlying mechanism using in vivo and in vitro models. The MFAP4-/- mice were subjected to unilateral ureteral obstruction (UUO) to elucidate the role of MFAP4 in renal fibrosis in vivo. Compared to the wild-type mice, the MFAP4-/- mice exhibited decreased protein expression of p-p65 and p-IKBα and ECM deposition after UUO. The MFAP4-/- mice exhibited attenuated nuclear translocation of p65 (the hub subunit of nuclear factor (NF)-κB signaling pathway), suppressed activation of transforming growth factor (TGF)-β/Smad pathways, and downregulated expression of fibronectin, collagen I, and plasminogen activator inhibitor-1. The knockdown of MFAP4 mitigated the TGF-β-induced upregulated expression of fibronectin, collagen I, and plasminogen activator inhibitor-1 in the human proximal tubular epithelial cells (HK-2). Compared to the HK-2 cells transfected with sh-MFAP4, the HK-2 cells co-transfected with sh-MFAP4 and Ad-MFAP4 exhibited severe inflammatory response and increased fibrosis-related proteins expression. Mechanistically, the knockdown of MFAP4 inhibited the activation of NF-κB and TGF-β/Smad signaling pathways and downregulated the expression of fibrosis-related proteins. The findings of this study indicate that MFAP4 is involved in UUO-induced renal fibrosis through regulation of NF-κB and TGF-β/Smad pathways.