In this study we aimed to investigate the role of tomato seed oil (TSO) in the alleviation of hyperlipidemia and the regulation of gut microbiota in C57BL/6J mice. Mice were divided into the following four diet-based groups: low-fat diet (LF, n = 8), high-fat diet (HF, n = 6), HF diet with TSO replacing one-third of lard (TL, n = 8), and HF diet with TSO replacing two-thirds of lard (TH, n = 8). The results showed that TH significantly reduced weight gain, relative adipose tissue weights, plasma cholesterol, triacylglycerol, low-density lipoprotein cholesterol (LDL-C), ratio of LDL-C to high-density lipoprotein cholesterol (HDL-C), hepatic cholesterol, and total fatty acids, and markedly increased plasma HDL-C. TSO supplementation also dose-dependently increased fecal cholesterol excretion and reduced fecal total fatty acids. This was accompanied by upregulation of the gene expression of hepatic PPARα, ACADL, CYP7A1, LXRα, ABCA1, and SR-B1. Metagenomic analyses demonstrated that TSO tended to reduce the Firmicutes/Bacteroidetes ratio, significantly increased the relative abundance of the genus Lactobacillus, and reduced the relative abundance of the genera Rikenella, Enterorhabdus, unclassified_o_Clostridiales and Ruminococcaceae_UCG-009. These results proved that TSO was effective in attenuating hyperlipidemia in C57BL/6J mice by enhancing fatty acid β-oxidation, reducing cholesterol absorption, promoting cholesterol efflux, and favorably modulating the gut microbiota.