Chlorpyrifos (CPF) is a class of toxic compounds which has been widely used in agriculture that can cause multi-organ damage to the liver, kidneys, testes, and nervous system. Currently, most studies on ginseng have concentrated on the roots and rhizomes, and less research has been conducted on the above-ground parts. Our laboratory found that ginseng stem and leaf total saponin (GSLS) features strong antioxidant activity. In this experiment, we selected different concentrations of CPF to induce hippocampal neuronal cell injury model in mice, conducted a cell survival screening test, and also selected appropriate concentrations of CPF to induce brain injury model in mice. CCK-8, flow cytometry, Elisa, Hoechst 33258 staining, Annexin V-FITC/PI staining, HE staining, Morris water maze, and qRT-PCR were adopted for detecting the effects of GSLS treatment on CPF-induced cell viability, mitochondrial membrane potential, reactive oxygen species (ROS) levels, Ca2+ concentration and GSLS treatment on CPF-induced brain injury and related signaling in mice, respectively. The effects of GSLS treatment on CPF-induced brain injury and the related signaling pathways in mice were examined. The results showed that GSLS at 60 μg/ml and 125 μg/ml concentrations elevated the viability of CPF-induced HT22 cells, increased mitochondrial membrane potential, depleted ROS, decreased Ca2+ concentration, and decreased apoptosis rate. Meanwhile, GSLS treatment significantly reduced CPF-induced escape latency in mice, elevated the number of entries into the plateau and effective area, increased the effective area and target quadrant residence time, as well as improved the pathological damage of mouse hippocampal neurons. The results of mouse brain sections demonstrated that GSLS treatment significantly increased SOD and CAT activities and lowered MDA accumulation in CPF-induced mice. qRT-PCR revealed that PTEN mRNA expression was significantly decreased with PI3K and AKT expression being significantly increased in GSLS-treated CPF-induced mice. Thus, the obtained results indicate that GSLS can effectively antagonize CPF-induced brain toxicity in mice through regulating PTEN/PI3K/AKT pathway.