Objective To investigate the early alterations of neurometabolites in the splenium of corpus callosum by proton magnetic resonance spectroscopy (~1H-MRS) in patients with diffuse axonal injury (DAI) at early stage and their prognostic value. Methods Twenty-one patients with DAI underwent 1H-MRS examination in the splenium of corpus callosum 2 to 14 d after injury, the neurometabolite alterations were evaluated and compared with those of 10 healthy subjects (normal controls). Logistic regression analysis was performed to explore the correlation among neurometabolite alterations, clinical indexes and Glasgow outcome scale (GOS) six months after injury. Results There was a significant decrease in NAA/Cr and NAA/Cho and increase in Cho/Cr in the splenium of corpus callosum in patients with DAI as compared with control group (P<0.05), and these trends tended to be more evident with the increase of injury severity. NAA/Cr and NAA/Cho in patients with poor outcomes were lower than those in patients with good outcomes (P<0.05). NAA/Cr, NAA/Cho and Cho/Cr predicted long-term outcome with 89% accuracy, and the combination with GOS provided the predictive accuracy of 94%. Conclusion ~1H-MRS examination in the splenium of corpus callosum at early stage of DAI can depict brain injury severity, and is useful in predicting outcomes.