Ganoderma lucidum polysaccharide (GLP) extracted from Ganoderma lucidum (Leyss. ex Fr.) Karst, a traditional Chinese medicine, is a biologically active substance reported to possess anti-oxidative, anti-apoptotic, and neurological protection. However, it is unknown whether GLP have any protective effect against high-fat constituents-induced epithelial cell injury. The aim of this study was to investigate the protection and molecular mechanism of GLP on injury induced by palmitic acid (PA) in the intestinal porcine epithelial cell line (IPEC-J2). First, we tested whether the treatment of GLP attenuate PA-induced IPEC-J2 cell death. GLP markedly blocked PA-caused cytotoxicity and apoptosis in IPEC-J2 cells. Moreover, GLP recovered the decreased mitochondrial function and inhibited activation of caspase-dependent apoptotic pathway. Interestingly, PA promoted cell apoptosis and autophagy through stimulation of phosphorylation of mitogen-activated protein kinases (MAPKs), AMP-activated protein kinase (AMPK), and inhibition of phosphorylation of Akt and mammalian target of rapamycin (mTOR), which was reversed by GLP. Taken together, this study revealed a protective effect of GLP against PA-evoked IPEC-J2 cell death through anti-apoptotic and anti-autophagic properties.