Objective To investigate the neuroprotective effect of 2,4-diamino-6-hydroxypyrimidine (DAHP) on rats with brain injuries and its possible mechanism. Methods SD rat models of brain trauma were prepared by the Feeney method. The rats were randomly divided into model, DAHP, NOS activator [ tetrahydrobiopterin (BH4)], and DAHP + BH4 groups with 12 rats in each group. Another 12 rats were used for the sham operation group that only underwent bone window opening without percussion. Two hours after modeling, the DAHP group was injected with 0. 5 g / kg DAHP through the caudal vein, the BH4 group was injected with 0. 3 mg / kg BH4 through the caudal vein, the DAHP+BH4 group was injected with 0. 5 g / kg DAHP and 0. 3 mg / kg BH4 through the caudal vein, and sham operation and model groups were injected with the same volume of normal saline at an injection volume of 10 mL/ kg once a day for 1 week. After the last treatment, rat behavior was assessed by positioning navigation and space exploration experiments. Hematoxylin-eosin staining was used to observe histomorphology of the rat brain. TUNEL staining was used to observe apoptosis of brain cells. The Griess method was used to measure the level of nitric oxide (NO) in brain tissue, and expression of neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase ( iNOS), nuclear factor-κB (NF-κB), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), Caspase-3, and B-lymphoma-2 (Bcl-2) in brain tissue was detected by Western blot. Results In the sham operation group, the morphology of nerve cells in brain tissue was normal and nuclei were stained evenly. In the model group, nerve cells had swelled, deformed, and underwent nuclear pyknosis, which were accompanied by inflammatory cell infiltration. Compared with the model group, the morphology of nerve cells in the DAHP group was improved to some extent and the degree of inflammatory cell infiltration was reduced. The number of necrotic nerve cells was increased and the degree of injury was aggravated in the BH4 group. Morphological changes of nerve cells in the DAHP+BH4 group were not significant. Compared with the sham operation group, escape latency was prolonged; the proportion of space exploration time and Bcl-2 protein in brain tissue were decreased; and the apoptosis rate, NO level, and nNOS, iNOS, NF-κB, TNF-α, COX-2, and Caspase-3 protein expression were increased in brain tissue of the model group (P< 0.05). Compared with the model group, escape latency was shortened; the proportion of space exploration time and Bcl-2 protein in brain tissue were increased; and the apoptosis rate, NO level, and nNOS, iNOS, NF-κB, TNF-α, COX- 2, and Caspase-3 protein expression in brain tissue were decreased in the DAHP group (P< 0.05). Compared with the model group, escape latency was prolonged; the proportion of space exploration time and Bcl-2 protein in brain tissue were decreased; and the apoptosis rate, NO level, and nNOS, iNOS, NF-κB, TNF-α, COX-2, and Caspase-3 protein expression in brain tissue were increased in the BH4 group (P< 0.05). The escape latency; apoptosis rate in brain tissue; NO level; and nNOS, iNOS, NF-κB, TNF-α, COX-2, and Caspase-3 protein expression in the DAHP+BH4 group was lower than those in the BH4 group and the proportion of space exploration time and Bcl-2 protein expression in brain tissue were higher than those in the BH4 group (P< 0.05). Conclusions DAHP alleviates neurological impairment and reduces neuronal apoptosis in rats with brain injuries and its mechanism may be related to inhibition of the iNOS signaling pathway.