Purpose <p>The in vivo processes underlying neural stem cell (NSC) transplantation for ischemic stroke remain unclear, primarily due to the lack of effective in vivo monitoring and evaluation approaches. Therefore, we systematically investigated the in vivo mechanisms of NSC transplantation by positron emission tomography (PET) on a rat model with ischemic stroke.</p> Methods <p>NSCs were generated from human induced pluripotent stem cells (hiPSCs), and their neuronal differentiation potential was assessed in vitro. After transplantation into rats with photothrombotic stroke, neurological function was evaluated using the cylinder test and forelimb placing test. ¹⁸F-FDG, ¹⁸F-SynVesT-1 and ¹⁸F-DPA-714 PET were used to monitor brain metabolism, synaptic density, and glial reactivity in vivo. And correlations among the results of in vivo experiments were analyzed. Neuronal differentiation, maturation and synaptic connection of transplanted NSCs were investigated, while changes of glial reactivity were explored.</p> Results <p>NSCs derived from hiPSCs exhibited high neuronal differentiation potential in vitro. After NSC transplantation, neurological function of rats with ischemic stroke was recovered, while transplanted NSCs differentiated into neurons and established synaptic connections in the rat brain. In vivo brain metabolism and synaptic density were both restored, revealed by ¹⁸F-FDG and ¹⁸F-SynVesT-1 PET imaging and highly correlated with behavioral recovery. In vivo glial reactivity was higher at 8 weeks after NSC transplantation in ¹⁸F-DPA-714 PET imaging, mainly induced by microglial M2 polarization, which promotes neurological function recovery.</p> Conclusion <p>Our findings suggest PET molecular imaging can systematically monitor the brain metabolism, synaptic density and glial reactivity, providing effective in vivo monitoring and evaluation approaches for NSC transplantation in ischemic stroke.</p>