Left-right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3-CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left-right asymmetry of hippocampal function in behaving mice, we analyzed the performance of “split-brain” mice in the Barnes maze. The “split-brain” mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory-driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two-choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non-spatial yet hippocampus-dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus. © 2010 Wiley Periodicals, Inc.