The walnut fruit diaphragm (WFD) is an economically and ecologically important by-product of the Juglandaceae family. Components in the ethanolic extract of WFD (E-WFD) were identified by gas chromatography-mass spectroscopy (GC-MS). In our study, the lipid-lowering effect, antioxidant potential, and anti-inflammatory properties of E-WFD in the blood, heart, and liver tissues of rats (♂, Wistar) with hyperlipidemia caused by Triton WR-1339 were determined using biochemical, histopathological, and molecular techniques. Experimental animals for analysis are as follows: K1: healthy control group, K2: E-WFD (150 mg, o.d.), K3: E-WFD (300 mg, o.d.), K4: HL (hyperlipidemic group) (400 mg kg–1 Triton WR-1339, i.p.), K5: HL + E-WFD (300 mg) grouped as (n = 6). After the application, anti-hyperlipidemic analysis (TC, TG, LDL-C, HDL-C, ALT, and AST), oxidative stress (MDA, SOD, and CAT) parameters measurement, histopathological examinations, and inflammatory biomarker genes (Tnf-α, Il-6, Il-1β, Mmp-2, Mmp-9, Lxr-α and Srebp-1c) expression stability were determined by q-RT PCR. With the E-WFD application, there was a decrease in TC and TG levels and an increase in HDL-C. Oxidative stress decreased by changing MDA, SOD, and CAT levels. Histopathological deterioration in the tissues changed positively. In addition, E-WFD showed significant inhibitory properties on Tnf-α, Il-6, Il-1β, Mmp-2, Mmp-9, Lxr-α, and Srebp-1c genes. Practical applications: It is known that hyperlipidemia causes a wide range of diseases. In this study, the effect of walnut fruit diaphragm (WFD) ethanol extract was compared against the effect of Triton WR-1339-induced hyperlipidemia on rats. With the use of WFD, the damages caused by hyperlipidemia were significantly ameliorated. Accordingly, it is expected to contribute to the pharmacology, food and cosmetic industries as a very valuable alternative product.].