Stroke remains a leading cause of global mortality and long-term disability. Chitosan-based nitric oxide nanoparticles (CS-NO), a novel injectable hydrogel, demonstrate protective effects in ischemia/reperfusion injury. However, the therapeutic efficacy and underlying mechanisms of CS-NO in stroke remain unclear. Here, we found that CS-NO exerted significant neuroprotective effects, as evidenced by neurofunctional assessments, along with marked reductions in neuronal apoptosis and inflammatory responses in stroke male mice models. Mechanistically, RNA-seq analysis combined with functional studies using an AAV9 delivery system revealed that the pathological interaction between HIF-1α and the MAPK pathway serves as a key driver in stroke development, which could be effectively inhibited by CS-NO administration. Notably, combination therapy involving CS-NO with either HIF-1α or TLR4 inhibitors showed superior neurological outcomes compared to monotherapy in stroke male mice. Collectively, CS-NO's neuroprotection may stem from disrupting HIF-1α-MAPK crosstalk following stroke, suggesting the need for translational studies to further validate its clinical potential. This study introduces a novel chitosan-based nitric oxide (NO)-releasing hydrogel (CS-NO) designed for responsive release in the ischemic microenvironment, offering a targeted and sustained therapeutic approach distinct from bolus NO donors.