Green tea has been consumed as the most popular drink in East Asia for centuries, and is believed to have a wide range of health benefits. L-Theanine, the major component of the free amino acids in green tea, has been reported to display neuronal protection and tumor inhibition in vitro, but its physiological effects on animal development and behavior remain elusive. In this report, we used Drosophila melanogaster, the fruit fly, as a model organism to investigate the physiological effects of L-theanine. Flies were fed with three different concentrations of theanine as a dietary supplement after eclosion, and were examined for a variety of physiological parameters at different time points. We found theanine treatment results in significantly increased locomotion and courtship ability, and decreased resistance against wet and dry starvation in males, but not in females. Furthermore, theanine application diminished UV tolerance in females, but not in males. However, we did not perceive distinguishable effect of theanine on animal development, life span, weight, and tolerance of heat and anoxia. This work represents the first comprehensive physiological investigation of L-theanine at the whole animal level, and shall shed light on the mechanistic study of theanine in the future.