Aging-correlated cognitive declines, including deficiencies in spatial orientation and memory, may reflect dysfunction in the hippocampus and medial entorhinal cortex (MEC). However, aging-related changes in MEC at the cellular and molecular levels remain unclear. In this study, we found fewer grid cells with reduced spatial stability in old mice. We compared gene expression profiles between young and old mice using 10x Genomics Visium technology. Among 1664 differentially expressed genes, we discovered a marker gene for subpopulation in MEC Layer III with decreased cell number with age. Silencing of these neurons in young mice impaired the spatial tuning of neurons in MEC and the spatial learning of a new platform location in the water maze task. These findings help us to understand the cellular and molecular changes in the MEC in healthy aging animals and the potential role of certain neuronal populations in MEC microcircuits during aging.