Reactive oxygen species (ROS) play an important role in cellular signaling as second messengers. However, studying the role of ROS in physiological redox signaling has been hampered by technical difficulties in controlling their generation within cells. Here, we utilize two inert components, a photosensitizer and light, to finely manipulate the generation of intracellular ROS and examine their specific role in activating dendritic cells (DCs). Photoswitchable generation of intracellular ROS rapidly induced cytosolic mobilization of Ca(2+), differential activation of mitogen-activated protein kinases, and nuclear translocation of NF-κB. Moreover, a transient intracellular ROS surge could activate immature DCs to mature and potently enhance migration in vitro and in vivo. Finally, we observed that intracellular ROS-stimulated DCs enhanced antigen specific T-cell responses in vitro and in vivo, which led to delayed tumor growth and prolonged survival of tumor-bearing mice when immunized with a specific tumor antigen. Therefore, a transient intracellular ROS surge alone, if properly manipulated, can cause immature DCs to differentiate into a motile state and mature forms that are sufficient to initiate adaptive T cell responses in vivo.