It had been reported that enriched environment was beneficial for the brain cognition, neurons and synapses in cortex and hippocampus. With diffusion tensor imaging (DTI), several studies recently found the trained-induced larger corpus callosum. However, the effect of enriched environment on the oligodendrocytes in corpus callosum has not been explored with the unbiased stereological methods. In current study, the effect of enriched environment on the total number of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) positive cells in middle-aged rat corpus callosum was investigated by means of immunohistochemical techniques and the unbiased stereological methods. We found that, when compared to standard rats, the spatial learning capacity of enriched-environment rats was significantly increased. The total number of the CNPase positive cells in the corpus callosum of enriched-environment middle-aged rats was significantly increased when compared to standard rats. The present study provided, to the best of our knowledge, the first evidence of environmental enrichment-induced increases in the total number of CNPase positive cells in the corpus callosum of middle-aged rats.