Cx3cr1-driven Cre recombinase (Cre) is a widely used genetic tool for enabling gene manipulation in microglia and macrophages. However, an in-depth analysis of the possible detrimental effects of Cre activity in microglia, surprisingly, remains missing. Here, we demonstrate an age-dependent sensitivity of microglia to Cx3cr1-Cre toxicity, wherein Cre induction, specifically in early postnatal microglia, is detrimental to microglial development, proliferation, and function. Tamoxifen (TAM)-induced Cre activity leads to microglial activation, type 1 interferon (IFN-1) signaling, and increased phagocytosis, causing aberrant synaptic pruning during the early postnatal period and anxious behavior at later age. The detrimental effects of Cre induction are caused by DNA-damage-induced toxicity in microglia and are limited to the early postnatal period, showing no detrimental effects in adult microglia. Thus, our study reveals an age-dependent vulnerability of microglia to Cre activity, thereby highlighting age dependency of Cre action, which could be especially applicable in the broader context of environment-responsive cell types.