Application of low-frequency ultrasound has been shown to enhance transdermal drug transport of large molecules such as insulin. In this study, we investigated the dependence of ultrasound-induced transdermal delivery of insulin on ultrasound parameters. Insulin was delivered in vivo to hairless rats using 20 kHz ultrasound applied over a range of ultrasound intensity, application time and pulse length. Change in blood glucose levels of the animals was monitored to assess insulin transport. The results showed a threshold below which no detectable changes in blood glucose level was observed for each ultrasound parameter. Moreover, our findings indicated that sonophoretic enhancement is dependent on energy dose and length of ultrasound pulse that is consistent with a cavitation-based mechanism. The more significant effect of lowering glycemia was obtained with application of less than 15 min ultrasound and was similar to subcutaneous injection of 0.5 U of insulin. Pretreatment of hairless rat skin with ultrasound followed by application of insulin resulted in no significant modification in blood glucose level, indicating that transdermal transport of insulin mainly occurred during sonication. Sonophoresis may therefore potentially be applied for non-invasive and painless delivery of insulin in the treatment of insulin-dependent diabetes.