It has been shown that 1 and 3 MHz low-intensity ultrasound was able to affect the fragile and leaky angiogenic blood vessels in a tumor. However, the biological effects of 21 kHz low-intensity ultrasound on tumors remain unclear. The aim of the present study was to explore the effects of 21 kHz ultrasound with microbubbles on the regulation of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2) and apoptosis in subcutaneous prostate tumors in nude mice. The study included three parts, each with 20 tumor-bearing nude mice. Twenty nude mice were divided into four groups: control (sham treatment), microbubble ultrasound contrast agent (UCA), low-frequency ultrasound (US) and US+UCA groups. The UCA used was a microbubble contrast agent (SonoVue). The parameter of ultrasound: 21 kHz, an intensity of 26 mW/cm2, 40% duty cycle (on 2 sec, off 3 sec), 3 min, once every other day for 2 weeks. In the first study, all subcutaneous tumors were examined by contrast-enhanced ultrasonography (CEUS) at the initiation and completion of the experiments. Peak intensity (PI), time to peak intensity (TTP) and area under the curve (AUC) on the time intensity curve (TIC) were analyzed. In the second study, the intensity of VEGF and COX-2 protein expression in the vascular endothelium and cytoplasm was evaluated using immunohistochemistry and laser confocal microscopy. In the third study, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay was used for the evaluation of cell apoptosis in tumor tissues. The tumor cells and vasculature were examined by transmission electron microscopy (TEM). Only in the US+UCA group, PI and AUC decreased. The intensity of COX-2 and VEGF in the US+UCA group in immunohistochemical staining and laser confocal microscopy was lower compared to that of the other three groups. More cell apoptosis was found in the US+UCA group compared to the other 3 groups. In the control, UCA and US groups, the tumors had intact vascular endothelium and vessel lumens in TEM. However, lumen occlusion of vessels was observed in the US+UCA group. Twenty-one kHz low-intensity ultrasound with microbubbles may have anti-angiogenic effects on subcutaneous tumors in nude mice.