Cholinergic cells originating from the medial septal nucleus (MS) and the vertical and horizontal limbs of the diagonal band of Broca (VDB and HDB, respectively) are critical for supporting a variety of memory and cognitive functions. However, the viability of cholinergic cells has not been explored in the context of acute hypoxia (AH). This study aimed to investigate the effects of AH on cholinergic cells in these nuclei and to test whether hypoxic preconditioning (HPC)-a previously established neuroprotective therapy-could prevent cholinergic cell loss, cognitive dysfunction, and hippocampal synaptic dysfunction in mice exposed to AH. We found that cholinergic cell loss occurred in the MS/DB after AH. HPC prevented this effect and also improved AH-induced cognitive dysfunction and hippocampal synaptic dysfunction. Overall, our findings highlight the significant role of cholinergic cells in AH-induced memory impairments and suggest that the preservation of cholinergic cell viability may provide a mechanism by which HPC improves memory impairments and preserves the function of memory-processing brain structures after AH.