Near-infrared (NIR) persistent phosphor ZnGa2O4:Cr3+ (ZGC) has unique deep-tissue rechargeable afterglow properties. However, the current synthesis leads to agglomerated products with irregular morphologies and wide size distributions. Herein, we report on in vivo rechargeable mesoporous SiO2/ZnGa2O4:Cr3+ (mZGC) afterglow NIR-emitting nanocomposites that are made by a simple, one-step mesoporous template method. At less than 600 °C, pores in mesoporous silica nanoparticles (MSNs) act as nanoreactors to generate in situ ZnGa2O4:Cr3+ NIR-persistent phosphors. The as-synthesized mZGC preserves defined size, morphology, and mesoporous nanostructure of the MSNs. The persistent luminescence of the as-synthesized mZGC is recharged in a simulated deep-tissue environment (e.g., ≈8 mm pork slab) in vitro by using red light (620 nm). Moreover, mZGC can be repeatedly activated in vivo for persistent luminescence imaging in a live mouse model by using white LED as a light source. Our concept of utilizing mesoporous silica as nanoreactor to fabricate ZGC PL nanoparticles with controllable morphology and preserved porous nanostructure paves a new way to the development and the wide application of deep tissue rechargeable ZGC in photonics and biophotonics.