Gynostrmma pentaphyllum seed oil (GPSO), extracted from G. pentaphyllum seeds, is rich in conjugated linolenic acid, which is a special fatty acid consisting of cis-9, trans-11, trans-13 isomers. Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia resulting from insulin resistance, and is usually accompanied by hypertension, hyperlipidemia, atherosclerosis (i.e., the metabolic syndrome, or syndrome X), and polycystic ovarian disease. This study aimed to investigate the effect of GPSO on T2DM hepatic lipid metabolism and the underlying mechanism involving level of protein expression. In the experiment, the model of T2DM was established. Kunming male mice were fed with a high-fat diet and injected with streptozocin, in which the exploration of detailed mechanism in the therapy of T2DM was targeted. The results showed that the ability of oral glucose tolerance was improved in the GPSO group. Biochemical indices also revealed that GPSO had a positive effect on hypoglycemic activity, suggesting that GPSO could promote the expression of glucose transporter 4 in liver and skeletal muscle.