L-Theanine is an amino acid ingredient in green tea with a structural analogy to glutamine and is suggested to be taken up into the cells via an L-glutamine transporter Slc38a1. Recently, the oral intake of L-theanine is expected to suppress anxiety, sleep disturbance, and cognitive impairment. Some reports showed that L-theanine possesses anticancer activities against some cancers. Here, it was investigated whether L-theanine inhibits cell proliferation and its mechanism is via Slc38a1. L-Theanine inhibited cell proliferation in mouse motor neuron cell line (NSC-34), mouse neuroblastoma cell line (Neuro 2A) and human neuroblastoma cell line (SH-SY5Y) in a dose- and time-dependent manner. However, it had little effect in human brain glioblastoma cell line (U-251 MG), mouse astrocyte cell line (C8-D1A), mouse brain endothelial cell line (bEnd3) and human umbilical vein endothelial cells (HUVEC). There was a positive correlation between the L-theanine-dependent inhibition of cell proliferation and the expression level of Slc38a1 mRNA (r² = ～0.6). Therefore, it was suggested that in these cell lines, the suppressive effect on cell proliferation was caused by L-theanine which was taken up into the cells via Slc38a1.