The escalating prevalence of metabolic dysfunction-associated fatty liver disease (MAFLD) poses a significant global health burden. MAFLD is characterized by abnormal lipid accumulation in liver parenchymal cells and activation of endoplasmic reticulum stress. However, the effect of Krüppel-like factor 16(KLF16) on glycolipid metabolism has been limited. We investigated whether KLF16 could alleviate MAFLD through endoplasmic reticulum stress.HepG2 and mouse primary hepatocytes were treated with oleic acid (OA) to model MAFLD in vitro. siRNA was used to downregulate KLF16 expression in cells. C57/BL6J mice were fed a high-fat diet to model MAFLD in vivo, and AAV8 was used to regulate KLF16 and ATF4 expression. Western-blot, RT-qPCR, oil red O staining, and dual-luciferase reporter assays were used to explore the underlying mechanisms in MAFLD. T-test and ANOVA were used to compare the differences among groups.KLF16 expression was upregulated in MAFLD models. KLF16 downregulation aggravates lipid deposition in liver cells. The expression of ATF4 protein was downregulated in MAFLD models with KLF16 knockdown. KLF16 transcriptionally regulates ATF4 expression through two binding sites. ATF4 overexpression alleviates lipid deposition exacerbated by KLF16 knockdown in mice.In the face of abnormal lipid deposition in MAFLD, liver cells can play a spontaneous protective role by upregulating KLF16, which can promote the expression of ATF4 at the transcriptional level, further affecting downstream lipid metabolism-related genes. KLF16 may serve as a promising target gene to improve the progression or prognosis of MAFLD.