Abstract Background The risk of developing Alzheimer’s disease (AD) is modulated by genetic and environmental factors. Early-life stress (ELS) exposure during critical periods of brain development can impact later brain function and health, including increasing the risk of developing AD. Microglial dysfunction and neuroinflammation have been implicated as playing a role in AD pathology and may be modulated by ELS. To complicate matters further, sex-specific effects have been noted in response to ELS and in the incidence and progression of AD. Methods Here, we subjected male and female mice with either a wild type or 5xFAD familial AD-model background to maternal separation (MS) from postnatal day 2 to 14 to induce ELS. Results We detected hippocampal neuroinflammatory alterations already at postnatal day 15. By 4 months of age, MS mice presented increased immobility time in the forced swim test and a lower discrimination index in the novel object recognition memory test compared to controls. We found altered Bdnf and Arc expression in the hippocampus and increased microglial activation in the prefrontal cortex due to MS in a sex-dependent manner. In 5xFAD mice specifically, MS exacerbated amyloid-beta deposition, particularly in females. In the periphery, the immune cell population was altered by MS exposure. Conclusion Overall, our results demonstrate that MS has both short- and long-term effects on brain regions related to memory and on the inflammatory system, both in the brain and periphery. These ELS-related effects that are detectable even in adulthood may exacerbate pathology and increase the risk of developing AD via sex-specific mechanisms.