Microglia cells are essential for brain homeostasis and have essential roles in neurodegenerative diseases. Aging is the main risk factor for most neurodegenerative diseases, and age-related changes in microglia may contribute to the susceptibility of the aging brain to dysfunction and neurodegeneration. We have analyzed morphology and dynamic behavior of neocortical microglia in their physiological environment in young adult (3-month-old), adult (11- to 12-month-old), and aged (26- to 27-month-old) C57BL/6J-Iba1-eGFP mice using in vivo 2-photon microscopy. Results show that surveying microglial cells in the neocortex exhibit age-related soma volume increase, shortening of processes, and loss of homogeneous tissue distribution. Furthermore, microglial process speed significantly decreased with age. While only a small population of microglia showed soma movement in adult mice, the microglia population with soma movement was increased in aged mice. However, in response to tissue injury, the dynamic microglial response was age-dependently diminished. These results provide novel insights into microglial behavior and indicate that microglial dysfunction in the aging brain may contribute to age-related cognitive decline and neurodegenerative diseases.