Oxidative stress damage of pancreatic β-cells is a key link in the pathogenesis of type 2 diabetes mellitus. A long-term increase of free fatty acids induces the increase of reactive oxygen species (ROS) in β-cells, leading to apoptosis and dysfunction of β-cells. Ganoderma lucidum spore oil (GLSO) is a functional food complex with strong antioxidant activity, but its solubility and stability are poor. In the present study, GLSO-functionalized selenium nanoparticles (GLSO@SeNPs) with high stability and uniform particle size were synthesized by a high-pressure homogeneous emulsification method. The aim of this study was to investigate the protective effects of GLSO@SeNPs on INS-1E rat insulinoma β-cells against palmitic-acid (PA)-induced cell death, as well as the underlying mechanisms. Our results showed that GLSO@SeNPs had good stability and biocompatibility, and they significantly inhibited the PA-induced apoptosis of INS-1E pancreatic cells by regulating the activity of related antioxidant enzymes, including thioredoxin reductase (TrxR), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px). Western blot analysis showed that GLSO@SeNPs reversed the PA-induced changes in MAPK pathway protein expression levels. Thus, the present findings provided a new theoretical basis for utilizing GLSO@SeNPs as a treatment for type 2 diabetes.