Objective To detect the content changes of endogenous hydrogen sulfide (H2S) in the cerebral tissues after diffuse axonal injury (DAI) and observe the role of exogenous H2S in DAI so as to clarify the role of H2S as a new gas signaling molecule involved in the process of DAI and provide rationale supports for the prevention and treatment of neurological dysfunction after DAI. Methods The DAI model was established by a coronal rotation device and exogenous H2S was administered for intervention. Time points of 6h, 24h and 7d after DAI were chosen for observation. Methylene blue spectrophotometric method was used for detecting H2S content change in the brain tissues after DAI, flow cytometry for measuring the apoptosis of hippocampal CA3 neurons, and Western blot for determining the change of β-amyloid precursor protein (β-APP) expression in the cortex, hippocampus and brain stem. Results Compared with that in the control group, endogenous H2S content in the cortex, hippocampus and brainstem significantly increased in DAI groups, with DAI+H2S intervention groups having significantly higher H2S content than DAI model groups. The apoptosis of hippocampal CA3 neurons showed an increased early apoptosis percentage in the DAI model groups and more apoptotic neuron cells were observed in the longer time post-injury in DAI model groups. Furthermore, DAI (7d) + H2S group had a significantly higher early apoptosis percentage than DAI (7d) group. More importantly, Western blot showed that β-APP in the cortex, hippocampus and brain stem in DAI model groups was expressed higher than in the control group, with DAI+H2S intervention groups having a significantly higher β-APP expression than DAI model groups. Conclusion The increase of neuronal apoptosis and β-APP expression may be related to the increase of endogenous H2S after DAI. It suggests that H2S may be involved in the secondary neuronal injury of DAI as a new neurotransmitter. [ABSTRACT FROM AUTHOR]