Persistent luminescent nanoparticles (PLNPs) have emerged as important nanomaterials for biological imaging as a result of complete avoidance of tissue auto-fluorescence. However, the imaging sensitivity and long-term in vivo imaging are still limited due to the persistent luminescence that is rapidly decayed in vivo after an ex vivo excitation. To address this limitation, in vivo activation of PLNPs is highly desired. Herein, we present a new strategy for the activation of PLNPs (SrAl₂O₄:Eu²⁺) by using soft X-ray excitation. Importantly, as the soft X-ray light source possesses the advantage of deep tissue penetration, the PLNPs can be reactivated in vivo through living tissue using soft X-ray excitation. Furthermore, X-ray/persistent luminescence dual-modal imaging can be achieved to empower this strategy with high sensitivity. Our results suggest that this new strategy of in vivo energy charging in PLNPs would bring new insights for deep tissue and long-term bioimaging in living animals, and provide new perspectives for persistent luminescence bioimaging and therapeutic applications.