The effect of aging on learning and memory has been intensively studied. However, the mechanisms underlying impairment of memory functions at middle age remains inexplicit. To address this question, we assessed the spatial working memory and long-term memory of middle-aged (16–18 months) rats with delayed alternation in T-maze and water maze task respectively. We observed a significant impairment of spatial working memory in middle-aged rats in delayed alternation in T-Maze task, while long-term spatial memory remained unchanged. To further explore possible mechanisms underlying this age-associated impairment of spatial working memory, we examined the activity of RhoA in the prefrontal cortex, dorsal hippocampus, dorsal striatum and sensorimotor cortex. We found that middle-aged rats showed a significant decrease in RhoA activity in dorsal striatum but not in other regions examined, while the protein level remained unchanged compared to the young rats (2–3 months). Moreover, we found that microinfusion of Y-27632, a specific inhibitor of the ROCK that is a downstream effector of RhoA, into dorsal striatum of young rats also impaired their working memory tested in delayed alternation in T Maze task. These results suggest that RhoA activity in dorsal striatum may play a role in mediating spatial working memory.