Microglia, the major brain immune cells, have multifaceted roles not only in maintaining immune homeostasis, but also in driving neurodevelopment, regulating satiety, promoting memory, and contributing to other non-immune functions. Many of these microglia-mediated functions are affected by circadian rhythms, fluctuating throughout the course of the day. Emerging evidence suggests microglia contribute to this circadian rhythmicity. To test the role of microglia in regulating circadian rhythms, we used our Cx3cr1-Dtr transgenic Wistar rat model to acutely deplete microglia and examined if this could lead to a disruption in circadian temperature, metabolism and activity measures. We also examined if shifts in these physiological rhythms correspond with changes in the expression of key circadian rhythm-regulating genes and proteins. Our data show that microglial depletion leads to a profound disruption of circadian rhythms in several domains consistent with a shift towards the inactive phase throughout the day and night. These shifts in circadian rhythmicity are accompanied by changes in the expression of central circadian rhythm-regulating genes and proteins. These findings indicate microglia are potential dynamic regulators of circadian rhythms in the CNS and indicate an exciting possibility to manipulate these cells to restore disrupted circadian rhythms such as with shift-work or jet-lag.