The hippocampus supports several mnemonic processes that may be critical to the accurate encoding and retrieval of spatial information, including pattern separation. Pattern separation is a mechanism for separating overlapping patterns of activation to distinguish similar patterns from one another. Several studies have shown that lesions of the dentate gyrus in rodents result in impaired spatial pattern separation for working memory and reference memory. Given the well-documented role of the dentate gyrus in supporting pattern separation and the susceptibility of this region to age-related changes, recent human studies have reported impaired pattern separation in older adults. Therefore, the current study investigated the age-related effects of the dentate gyrus on spatial pattern separation in a rodent model of aging. Fischer/Brown Norway (F344/BN) rats 6 months (n = 18) and 24 months (n = 18) of age were randomly assigned to an adjacent or a separate condition and tested on a place learning task using a radial eight-arm maze. Rats were trained to discriminate between a reward arm and a non-reward arm that either were adjacent to one another (high interference/ high pattern separation) or separated by a distance of two arm positions (low interference/ low pattern separation). Each rat received ten trials per day and was tested until the animal reached a criterion of nine correct choices out of ten consecutive trials across two consecutive days of testing. The results revealed no significant differences between young and aged rats in the number of trials required to reach the learning criterion in the adjacent or separate condition. In addition, rats in the adjacent condition required significantly more trials to reach the learning criterion than rats in the separate condition. However, an examination of the total errors committed before reaching the learning criterion revealed that aged rats make significantly more errors than young rats. In addition, more errors were committed in the adjacent condition than the separate condition. In the adjacent condition, there is high overlap among the external cues associated with the non-reward and reward arms, thus, potentially requiring more pattern separation. In contrast, there likely is less interference among the external cues in the separate condition resulting in less need for pattern separation. Our results are consistent with recent findings revealing impaired spatial pattern separation in older humans. These findings provide evidence for age-related changes in spatial pattern separation in older humans and animals.