The morphology and acoustic properties of the human umbilical vein endothelial cells (HUVECs) were evaluated using a scanning acoustic microscope system. HUVECs were cultured for 4 days and exposed to the endotoxin for 4 h. The frequency of the scanning acoustic microscope was variable between 100 and 210 MHz. By changing the measuring frequency, ultrasonic amplitude and phase were measured and the quantitative value of attenuation was calculated. Before and after endotoxin stimuli, HUVECs were observed by scanning acoustic microscopy and the attenuation was measured. The acoustic images were successfully obtained to identify the outer shape of the HUVEC and the location of the nucleus in the cell. The attenuation of the nucleus is higher than that of the cytoplasm. The attenuation of the cytoplasm was increased and became inhomogeneous after endotoxin exposure. This finding would be related to the change of F-actin filaments, which is the main component of the cytoskeleton. Scanning acoustic microscopy is useful for assessing the cellular viscoelastic properties since it can detect both the morphological and acoustic changes without contacting the cellular surface.