Aim To observe the effect of intraperitoneal injection of hydrogen-rich saline (HRS) on reverse cholesterol transport in golden hamsters with high fat diet. Methods The male golden hamsters were divided into three groups:chow diet group, high-fat diet group, and high-fat diet＋HRS group. After 12 weeks of high-fat diet feeding and HRS administration, macrophages labeled with ³H-cholesterol were injected into hypercholesterolemic golden hamsters, and then radioactivity in blood, liver, bile, and feces was measured. RT-qPCR and Western blot were used to assess the transcription and protein expression levels of cholesterol reverse transport-related genes in liver tissue. Results Long-term feeding with a high-fat diet induced significant hyperlipidemia and liver lipid accumulation in golden hamsters. Compared with the high-fat diet group, after HRS intervention, the body mass of golden hamsters decreased (P＜0.01), plasma TC and LDLC significantly decreased (P＜0.05), TG slightly decreased (P＝0.11), HDLC significantly increased (P＜0.01), oxidative stress index MDA in plasma and liver significantly decreased (P＜0.05 or P＜0.01), antioxidant index glutathione (GSH) significantly increased (P＜0.01), liver weight/body weight ratio slightly decreased (P＝0.05), TC and TG in liver decreased by 10.8% (P＝0.05) and 20.1% (P＜0.01), respectively. Liver steatosis was significantly relieved, but there was no significant change in inflammatory factor levels. In isotopic tracing, high-fat diet fed golden hamsters treated with HRS showed decreased ³H radioactivity in plasma at 24 and 48 hours by 16.5% (P＜0.01) and 8.9% (P＜0.05) respectively, while increased ³H radioactivity was observed in liver, bile, and feces by 1.2-fold (P＜0.05), 1.2-fold (P＝0.08), and 1.1-fold (P＝0.08) respectively, compared to those fed a high-fat diet alone. Furthermore, RT-qPCR and Western blot analyses of liver tissue demonstrated that HRS intervention resulted in a decrease of CD36, scavenger receptor-B1 (SR-B1), and low density lipoprotein receptor (LDLR) protein levels by 39.5% (P＜0.05), 40.5% (P＜0.01), and 28.0% (P＜0.01) respectively, an increase of ATP-binding cassette transporter G5 (ABCG5) and sterol regulatory element-binding protein (SREBP2) protein levels by 1.5-fold (P＜0.05) and 1.3-fold (P＜0.01), and an increase of mRNA levels of ATP-binding cassette transporter A1 and G8 by 2.9-fold (P＜0.05) and 3.2-fold (P＜0.01) respectively in high-fat diet-fed hamsters. Conclusions Intraperitoneal injection of HRS promotes reverse cholesterol transport in high-fat diet-fed golden hamsters and exerts lipid-lowering effects. Additionally, intraperitoneal injection of HRS may alleviate hepatic lipid accumulation by inhibiting hepatic lipid uptake and promoting cholesterol excretion from liver.