Sex differences in cognitive processing and function have been documented in human and animal studies. Females have been found to perform better than males on non-spatial memory tasks, while males tend to outperform females on spatial memory tasks. The neural mechanisms underlying these sexual dimorphisms are unclear. However, it is known that the cholinergic system is critically involved in memory processes, and there are notable differences between males and females in cholinergic system function and receptor expression. In particular, there are sex differences in the processing of information in the frontal cortex and hippocampus. In this study, we examined the roles of muscarinic and nicotinic acetylcholine receptors in the medial frontal cortex (MfC) and ventral hippocampus (VH) on spatial working memory in male and female rats. Local infusions of scopolamine (SCOP) and mecamylamine (MEC) (10, 20, 50 μg/side) were used to antagonize these receptors in each respective brain region during performance in the 16-arm radial arm maze. Infusions of SCOP into the VH caused a significant increase in memory errors in female rats, but had no significant effect on males, while infusions of MEC into the VH had no effect on either sex. Infusions of both SCOP (50 μg/side) and MEC (20 μg/side) into the MfC caused working memory impairments in both sexes. These results show that muscarinic acetylcholine receptors in the VH are differentially vulnerable to spatial memory impairments in females. Ventral hippocampal muscarinic acetylcholine receptors may play a key role in male-female differences in spatial memory.