Lactoferrin is present in brain cells and is known to protect against injury through anti-inflammatory and immunomodulatory processes. Furthermore, it has also been found within amyloid plaques in Alzheimer’s disease (AD), leading to the hypothesis that its presence in the AD brain may be detrimental. However, the identity of the brain cells that produce lactoferrin and how its expression changes in AD remain unclear. In this study, we examined lactoferrin expression and localization in 3- and 12-month-old male and female APP/PS1 mice. Using a microglial enrichment protocol and confocal imaging, we identified microglia as an important source of brain lactoferrin, and demonstrated that its expression is modulated by age, sex, and amyloid burden. In female mice, microglial lactoferrin expression was increased at both the mRNA and protein levels in 3-month-old APP/PS1 mice compared with age-matched wild-type controls. Conversely, lactoferrin transcript in the enrichment fraction, and protein localization were also reduced in microglia from 12-month-old female APP/PS1 mice, coinciding with increased amyloid burden. We further demonstrated that gene expression of lactoferrin follows daily variations under a light–dark cycle, similar to established microglial markers such as Iba1 and P2ry12, with alterations in this rhythmic pattern in APP/PS1 mice occurring predominantly in females. In addition, our findings suggest that neurons may internalize lactoferrin via LRP1, whose expression is downregulated by amyloid burden. This Aβ-dependent relationship was further corroborated in both microglial and neuronal in vitro models. Collectively, these findings identify microglia as an important source of lactoferrin in the brain. We suggest that sex- and age-dependent alterations in microglial lactoferrin expression may contribute to the sexual dimorphism observed in AD, possibly through alterations in LRP1-mediated neuronal uptake.