Non-invasive assessment of white-matter functionality in the nervous system would be a valuable basic neuroscience and clinical diagnostic tool. Using standard MRI techniques, a visual-stimulus-induced 27% decrease in the apparent diffusion coefficient of water perpendicular to the axonal fibers (ADC⊥) is demonstrated for C57BL/6 mouse optic nerve in vivo. No change in ADC|| (diffusion parallel to the optic nerve fibers) was observed during visual stimulation. The stimulus-induced changes are completely reversible. A possible vascular contribution was sought by carrying out the ADC⊥ measurements in hypercapnic mice with and without visual stimulus. Similar effects were seen in room-air-breathing and hypercapnic animals. The in vivo stimulus-induced ADC⊥ decreases are roughly similar to literature reports for ex vivo rat optic nerve preparations under conditions of osmotic swelling. The experimental results strongly suggest that osmotic after-effects of nerve impulses through the axonal fibers are responsible for the observed ADC decrease.