Objective: To observe the effects of oxymatrine on endoplasmic reticulum (ER) stress signaling pathway molecules ATF4, ATF6, IRE1 alpha, and fibroblast growth factor 21 (FGF21) as well as insulin resistance in high-fat diet rats. Methods: A total of 64 adult male SD rats were randomly divided into 8 groups. They were normal control group (CON), oxymatrine group (OM), high-fat diet group (HFD), high-fat diet + pioglitazone group (HFD+PZ), high-fat diet + low-dose oxymatrine (50 mg/kg) group (HFD+OM-L), high-fat diet + medium-dose oxymatrine (100 mg/kg) group (HFD+OM-M), and high-fat diet + high-dose oxymatrine (150 mg/kg) group (HFD+ OM-H) and high-fat diet + low-dose oxymatrine + pioglitazone group (HFD+OM+PZ), with 8 rats in each dose group. After 10-week intervention, the rats were abstained from food except water for 12 hours, they were weighed and then killed after being collected blood from abdominal aorta. The serum of rats in each group was separated for immediate measurement of fasting plasma glucose (FPG), fasting insulin (FINS), total cholesterol (TC) and triglyceride (TG). The homeostasis model of assessment for insulin resistance (HOMA-IR) was calculated. Meanwhile, the liver of rats was separated to measure the ATF4, ATF6, IRE1 alpha and FGF21 mRNA levels and their protein expression levels. The mechanism of oxymatrine on above endoplasmic reticulum stress pathway on insulin resistance pathway was also investigated. Results: Compared with CON, there was no significant difference in serum TG, TC, and FPG levels and HOMA-IR (P>0.05); at the same time, there was no difference in liver ATF4, AFT6, IRE1 alpha and FGF21 mRNA and protein levels (P>0.05) in OM. Compared with CON, serum TG, TC, and FPG levels as well as HOMA-IR increased obviously in HFD (P<0.05); meanwhile, ATF4, ATF6, IRE1 alpha mRNA levels and their protein expression levels apparently up-regulated, while FGF21 mRNA level and its protein level down-regulated (P<0.05). Compared with HFD, serum TG, TC, and FPG levels as well as HOMA-IR decreased obviously, ATF4, ATF6, IRE1 alpha mRNA levels and their protein expression levels in the liver apparently down-regulated, while FGF21 mRNA level and its protein level up-regulated (P<0.05) in HFD+ OM; no difference was found among different doses of oxymatrine (P>0.05). Compared with HFD, serum TG, TC, and FPG levels as well as HOMA-IR decreased obviously, meanwhile, ATF4, ATF6, IRE1 alpha and FGF21 mRNA levels and their protein expression levels apparently up-regulated (P<0.05) in HFD+ PZ. Compared with HFD, serum TG, TC, and FPG levels as well as HOMA-IR decreased obviously, meanwhile, ATF4, ATF6, IRE1 alpha mRNA levels and their protein expression levels in the liver were down regulated, and FGF21 mRNA level apparently up-regulated (P<0.05) in HFD+PZ+OM. Compared with HFD+ OM, there was no difference in serum TG, TC, and FPG levels as well as the decrease of HOMA-IR (P>0.05), meanwhile, no difference was found in ATF4, ATF6, IRE1 alpha and FGF21 mRNA levels and their protein expression levels in HFD+PZ+OM (P>0.05). Conclusion: Oxymatrine can not only down-regulate ATF4, ATF6, and IRE1 alpha mRNA levels and their protein levels, but also up-regulate FGF21 mRNA level and its protein level, besides it plays an important role in regulating the metabolism of glucose and insulin resistance in rats with hyperlipidemia.