This study explored the effect of Arc and its correlation with the impairment of spatial learning and memory in aged CD-1 mice. Ten CD-1 mice (5 males and 5 females) were selected from 3- and 15-month old respectively. The abilities of spatial learning and memory were accessed with Morris water maze (MWM), and the relative content of synaptic protein Arc in hippocampus were detected using immunohistochemical method. The results indicated that the swimming distance during the learning phase was significantly longer and the percentage of distance during the probe phase was significantly less in the 15-month mice than those in the 3-month old mice (F_(1,16)= 5.338, P < 0.05; F_(1,16)=78.013, P < 0.01). The 15-month old mice had significantly higher Arc in the CA1 and CA3 subregions of hippocampus than the 3-month mice (P < 0.05). In the mice aged at 15-month old, the Arc level in the CA1, CA3 and DG positively correlated with the learning swimming distance (P < 0.05), and the DG and CA3 negatively correlated with the probe percentage of distance within the target quadrant (P < 0.05). Our findings suggested that the Arc level of hippocampus increased in a subregion-specific manner, which might be involved in the impairment of spatial learning and memory in middle-aged CD-1 mice.