Several findings suggest a functional and anatomical differentiation along the dorso-ventral axis of the hippocampus. Lesion studies in rats have indicated that the dorsal hippocampus preferentially plays a role in spatial learning and memory, while the ventral hippocampus is involved in anxiety-related behaviors. Based on such findings our aim was to investigate the molecular differentiation along the dorso-ventral axis of the hippocampal granular cell layer of the rat dentate gyrus. Homogeneous isolation of this specific area was performed by laser-capture microdissection and Illumina microarray chips were used to identify genes differentially expressed in dorsal and ventral granular cell layer, respectively. Selected genes were confirmed by quantitative polymerase chain reaction analyses. From the total amount of 22518 probes 229 genes were found to be differentially expressed between dorsal and ventral granular cell layer with a false discovery rate below 5% and with a relative change in gene expression level of 20% or more. From this pool of genes 45 genes were more than two-fold regulated, 13 genes being dorsally enriched and 32 genes being ventrally enriched. Moreover, cluster analysis based on all genes represented on the microarray chip showed a clear differentiation between dorsal and ventral subgroups. Our findings demonstrate a dorso-ventral differentiation in gene expression even at the subregional level of the rat hippocampus, more specifically in the granular cell layer, substantiating the existence of functional heterogeneity along the dorso-ventral axis of the hippocampus.