Cancer stem cells play crucial roles in the development of cancer chemoresistance. L-Theanine, a nonproteinogenic amino acid derived from green tea, is gaining more and more attentions in reversing cancer drug resistance. However, its roles in development of lung cancer chemoresistance is still unknown. To investigate the effects of L-Theanine on chemoresistance and explore its underlying mechanism in lung cancer, we performed a panel of experiments in vitro combined with RNA-seq analysis and demonstrated L-Theanine improved the chemoresistance to cisplatin (cis-diamminedichloroplatinum; DDP) and inhibited stemness of DDP-resistant lung cancer cells but not non-resistant lung cancer cells and STAT3/NOTCH1 signaling was a potential dominant process involved in L-Theanine improving chemoresistance in DDP-resistant lung cancer. Mechanistically, L-Theanine impedes DDP-resistant lung cancer cells migration and activation via regulating the expression of STAT3/NOTCH1/BMAL1 signaling-induced stemness markers, reducing the migration and proliferation of DDP-resistant lung cancer cells as well as inhibiting drug resistance-related genes expression in DDP-resistant lung cancer cells. In addition, combination of L-Theanine and Stat3 blockade improved synergistically the chemoresistance in DDP-resistant lung cancer. In summary, L-Theanine improved chemoresistance in chemoresistant lung cancer through regulating STAT3/NOTCH1/BMAL1 signaling, reducing stemness and finally inhibiting the migration of DDP-resistant lung cancer cells. The finding might provide the research evidence for therapeutic options of reversing chemoresistance in lung cancer.