Prion protein peptide (PrP) has demonstrated neurotoxicity in brain cells, resulting in the progression of prion diseases with spongiform degenerative, amyloidogenic, and aggregative properties. Thymosin beta 4 (Tβ4) plays a role in the nervous system and may be related to motility, axonal enlargement, differentiation, neurite outgrowth, and proliferation. However, no studies about the effects of Tβ4on prion disease have been performed yet. In the present study, we investigated the protective effect of Tβ4against synthetic PrP (106–126) and considered possible mechanisms. Hippocampal neuronal HT22 cells were treated with Tβ4and PrP (106–126) for 24 h. Tβ4significantly reversed cell viability and reactive oxidative species (ROS) affected by PrP (106–126). Apoptotic proteins induced by PrP (106–126) were reduced by Tβ4. Interestingly, a balance of neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor) and receptors (nerve growth factor receptor p75, tropomyosin related kinase A and B) were competitively maintained by Tβ4through receptors reacting to PrP (106–126). Our results demonstrate that Tβ4protects neuronal cells against PrP (106–126) neurotoxicity via the interaction of neurotrophic factors/receptors.