Although it is known that injury enhances the regulatory activity of CD4 regulatory T cells (Tregs), the cellular and molecular mechanisms responsible for injury-induced Treg activation remain unclear. This study was designed to investigate and compare injury-induced T-cell receptor (TCR) signaling in Tregs, non-Tregs, and CD8 T cells. Specifically, we used phospho-flow cytometry to measure the expression and phosphorylation of ZAP-70, protein kinase C θ, nuclear factor of activated T cells, and glycogen synthase kinase 3β in FoxP3 Tregs versus FoxP3 non-Tregs versus CD8 T cells. Groups of male C57BL/6J mice underwent burn or sham injury, and lymph nodes and spleens were harvested at early time points-15, 30, 60, 120, and 240 min-to measure TCR signaling. As early as 15 min after burn injury, we observed a significant upregulation and phosphorylation of ZAP-70, protein kinase C θ, nuclear factor of activated T cells, and glycogen synthase kinase 3β in Tregs prepared from injury-site-draining lymph nodes. Burn injury did not activate TCR signaling in Tregs from the spleen or in CD4 non-Tregs and CD8 T cells. In conclusion, the results of this study demonstrate that burn injury activates TCR signaling in Tregs, but not non-Tregs or CD8 T cells. These findings suggest that injury provides an early TCR-activating signal to Tregs and supply new insights into how injury influences the adaptive immune system.