Previous studies have demonstrated that low intensity pulsed ultrasound (LIPUS) may accelerate wound healing and activate the proliferation of skin cells. Those studies however did not thoroughly explore the bioeffect with respect to ultrasonic frequency of insonations. Hence, experiments were arranged and carried out to extensively investigate the proliferation of human foreskin fibroblasts (HFFs) insonated for 3 minutes daily with various 20% pulsed ultrasounds of 1 kHz repletion frequency, fixed 200 mW/cm2 (SATA), and frequencies of 0.5, 1, and 3 MHz, within a period of 3 days. The viability of insonified HFFs was sampled and evaluated for every 12 hours by trypan blue staining and optical microscopy. Results showed that the rates of HFFs proliferation associated with insonations by both 1 and 3 MHz are larger than those of control groups. However, those of HFFs insonated with a 0.5 MHz ultrasound tend to be smaller than that of control groups. The timing corresponding to the largest rate of HFFs proliferation was found to vary with the frequency of insonation, in which it corresponds to 36 h and 24 h associated with insonations of 1 and 3 MHz ultrasound, respectively. Specifically, the insonations of HFFs with 1 and 3 MHz tend to activate the largest proliferation rates to be 1.5 and 1.3 fold, respectively, larger than that of the control group. The temperature in the culture plate was measured before and after ultrasonic insonation and that varied only within 1 oC implying that the activation of HFFs proliferation is associated with the non-thermal effect of ultrasonic stimulation.