Objective: To examine the effect of icariin (ICA) on the cognitive impairment induced by traumatic brain injury (TBI) in mice and the underlying mechanisms related to changes in hippocampal acetylation level. Methods: The modified free-fall method was used to establish the TBI mouse model. Mice with post-TBI cognitive impairment were randomly divided into 3 groups using the randomised block method (n=7): TBI (vehicle-treated), low-dose (75 mg/kg) and high-dose (150 mg/kg) of ICA groups. An additional sham-operated group (vehicle-treated) was employed. The vehicle or ICA was administrated by gavage for 28 consecutive days. The Morris water maze (MWM) test was conducted. Acetylcholine (ACh) content, mRNA and protein levels of choline acetyltransferase (ChAT), and protein levels of acetylated H3 (Ac-H3) and Ac-H4 were detected in the hippocampus. Results: Compared with the sham-operated group, the MWM performance, hippocampal ACh content, mRNA and protein levels of ChAT, and protein levels of Ac-H3 and Ac-H4 were significantly decreased in the TBI group (P＜0.05). High-dose of ICA significantly ameliorated the TBI-induced weak MWM performance, increased hippocampal ACh content, and mRNA and protein levels of ChAT, as well as Ac-H3 protein level compared with the TBI group (P＜0.05). Conclusion: ICA improved post-TBI cognitive impairment in mice by enhancing hippocampal acetylation, which improved hippocampal cholinergic function and ultimately improved cognition.