Tea (Camellia sinensis L.) is one of the most consumed beverages worldwide, not only due to its pleasant organoleptic properties, but also to its antioxidant effects. The amino acid L-theanine, which is almost exclusively found in tea leaves, has demonstrated a protective role against oxidative damages in certain cell types. We hypothesized that L-theanine could maintain the in-vitro redox homeostasis of human Sertoli cells (hSCs). The proper functioning of these cells is essential for a successful sperm production. So, we evaluated the effect of L-theanine (50 μM) on the metabolism, mitochondrial functionality, and oxidative damages in hSCs. The exposure of hSCs to L-theanine resulted in an increased hSCs proliferation and a subsequent increase in glucose metabolization through glycolysis. In response to this metabolic adaptation, there was an increase in mitochondrial membrane potential, which could result in a redox imbalance. However, no alterations were observed in protein carbonylation, nitration or lipid peroxidation relative to the non-exposed group. Our findings suggest that L-theanine (50 μM) can modulate hSCs function by sustaining the pools of Krebs cycle intermediates and by protecting cells from oxidative damage.