Generating spatially controlled, non-destructive changes in the interstitial spaces of the brain has a host of potential clinical applications, including enhancing the delivery of therapeutics, modulating biological features within the tissue microenvironment, altering fluid and pressure dynamics, and increasing the clearance of toxins, such as plaques found in Alzheimer's disease. Recently we demonstrated that ultrasound can non-destructively enlarge the interstitial spaces of the brain ex vivo. The goal of the current study was to determine whether these effects could be reproduced in the living brain using non-invasive, transcranial MRI-guided focused ultrasound (MRgFUS). The left striatum of healthy rats was treated using MRgFUS. Computer simulations facilitated treatment planning, and targeting was validated using MRI acoustic radiation force impulse imaging. Following MRgFUS treatments, Evans blue dye or nanoparticle probes were infused to assess changes in the interstitial space. In MRgFUS-treated animals, enhanced dispersion was observed compared to controls for 70 nm (12.8 ± 0.9 mm3 vs. 10.6 ± 1.0 mm3, p = 0.01), 200 nm (10.9 ± 1.4 mm3 vs. 7.4 ± 0.7 mm3, p = 0.01) and 700 nm (7.5 ± 0.4 mm3 vs. 5.4 ± 1.2 mm3, p = 0.02) nanoparticles, indicating enlargement of the interstitial spaces. No evidence of significant histological or electrophysiological injury was identified. These findings suggest that transcranial ultrasound can safely and effectively modulate the brain interstitium and increase the dispersion of large therapeutic entities such as particulate drug carriers or modified viruses. This has the potential to expand the therapeutic uses of MRgFUS.