Ultrasound-targeted microbubble (MB) destruction (UTMD) has been shown to increase the glomerular permeability, providing a potential novel therapeutic approach in targeted drug release for kidney diseases. Therefore, we investigated the impact of UTMD on renal interstitial permeability using MB-mediated diagnostic ultrasound (DUS). The left kidney of Sprague-Dawley (SD) rat was insonated by UTMD with either continuous or intermittent mode for 5 min. Evans blue (EB) revealed that both modes induced renal vascular permeability increase after DUS but recovered after 24 h. Intermittent insonation caused more severe injury than continuous mode. Red blood cells leaked out of the capillaries into interstitium without glomerular capillary hemorrhage (GCH) by hematoxylin and eosin (HE) staining. Electronic microscopy revealed the disruption of focal capillary wall in interstitial tissues. Morphological results confirmed capillary wall recovered in 24 h post-treatment. Results from fluorescence-labeled MBs showed that MBs were mainly localized in the interstitial portion of the tubular region and retained at 24 h. Intriguingly, urinalysis showed no clinical proteinuria after treatment. Our results indicated that MB plus DUS specifically and reversibly enhanced the interstitial permeability without affecting glomerulus, which may be developed into a therapeutic approach for targeting drug release to individual renal compartments.