Abstract Thymosin alpha-1 (Tα1) not only possesses immunoregulatory properties in periphery but also is expressed in the central nervous system (CNS) and affects the function of the CNS. To further elucidate the role of Tα1 in the CNS, the whole-cell recording technique was used to observe the effect of Tα1 on the spontaneous excitatory synaptic transmission in cultured rat hippocampal neurons. The results showed that acute treatment with Tα1 significantly enhanced the frequency of AMPA-mediated spontaneous excitatory postsynaptic current (sEPSC) at the concentrations of 1 and 10 μg/ml and also enhanced the frequency of AMPA-mediated miniature excitatory postsynaptic current (mEPSC) at 10 μg/ml. However, the amplitude of both sEPSC and mEPSC were not changed by Tα1. Those results suggested that Tα1 involves in the regulation of excitatory synaptic transmission in hippocampal neurons, which contribute to its neurophysiological function in the CNS.