Background: We recently reported a role for the circadian rhythm protein Period 2 (PER2) in midazolam induced cognitive dysfunction. Based on previous studies showing a critical role for the adenosine A2B receptor (ADORA2B) in PER2 regulation, we hypothesized that hippocampal ADORA2B is crucial for cognitive function. Methods: Midazolam treated C57BL/6J mice were analyzed for Adora2b hippocampal mRNA expression levels, and spontaneous T-maze alternation was determined in Adora2b-/- mice. Using the specific ADORA2B agonist BAY-60-6583 in midazolam treated C57BL/6J mice, we analyzed hippocampal Per2 mRNA expression levels and spontaneous T-maze alternation. Finally, Adora2b-/- mice were assessed for mRNA expression of markers for inflammation or cognitive function in the hippocampus. Results: Midazolam treatment significantly downregulated Adora2b or Per2 mRNA in the hippocampus of C57BL/6J mice, and hippocampal PER2 protein expression or T-maze alternation was significantly reduced in Adora2b-/- mice. ADORA2B agonist BAY-60-6583 restored midazolam mediated reduction in spontaneous alternation in C57BL/6J mice. Analysis of hippocampal Tnf-α or Il-6 mRNA levels in Adora2b-/- mice did not reveal an inflammatory phenotype. However, C-fos, a critical component of hippocampus-dependent learning and memory, was significantly downregulated in the hippocampus of Adora2b-/- mice. Conclusion: These results suggest a role of ADORA2B in midazolam induced cognitive dysfunction. Further, our data demonstrate that BAY-60-6583 treatment restores midazolam induced cognitive dysfunction, possibly via increases of Per2. Additional mechanistic studies hint towards C-FOS as another potential underlying mechanism of memory impairment in Adora2b-/- mice. These findings suggest the ADORA2B agonist as a potential therapy in patients with midazolam induced cognitive dysfunction.