The association of a sensory cue and an aversive footshock that are separated in time, as in trace fear conditioning, requires persistent activity in prelimbic cortex during the cue-shock interval. The activation of muscarinic acetylcholine receptors has been shown to facilitate persistent firing of cortical cells in response to brief stimulation, and muscarinic antagonists in the prefrontal cortex impair working memory. It is unknown, however, if the acquisition of associative trace fear conditioning is dependent on muscarinic signaling in the prefrontal cortex. Here, we delivered the muscarinic receptor antagonist scopolamine to the prelimbic cortex of rats prior to trace fear conditioning and tested their memories of the cue and training context the following day. The effect of scopolamine on working memory performance was also tested using a spatial delayed non-match to sample task. Male and female subjects were included to examine potential sex differences in the modulation of memory formation, as we have previously observed for pituitary adenylate cyclase-activating polypeptide signaling in the prefrontal cortex (Kirry et al., 2018). We found that pre-training administration of intra-prelimbic scopolamine impaired the formation of cued and contextual fear memories in males, but not females at a dose that impairs spatial working memory in both sexes. Fear memory formation in females was impaired by a higher dose of scopolamine and this impairment was gated by estrous cycle stage: scopolamine failed to impair memory in rats in the diestrus or proestrus stages of the estrous cycle. These findings add to the growing body of evidence that the prefrontal cortex is sexually dimorphic in learning and memory and additionally suggest that males and females differentially engage prefrontal neuromodulatory systems in support of learning.