Lhx5 is a member of the LIM homeobox gene family that regulates development of the nervous system. Adult mice generated with a mutation in Lhx5 were found to display absent or disorganized hippocampal neuroanatomy. The pyramidal cell layer in Ammon's horn and the granule cell layer in the dentate gyrus were absent or poorly defined in the hippocampus of adult Lhx5 knockout mice. Behavioral phenotyping of Lhx5 null mutants detected deficits on learning and memory tasks, including the Barnes maze spatial learning task, spontaneous alternation recognition memory, and contextual and cued fear conditioning. General health, neurological reflexes, and sensory abilities appeared to be normal in Lhx5 knockout mice. Motor tests showed impaired performance on some measures of motor activity, coordination, balance, and gait. These results reveal functional outcomes of Lhx5 gene deletion on the integrity of hippocampal neuroanatomy and behavior in the adult mouse.