The mechanical properties of cells have been verified to correlate to human diseases and aging as well as to various cellular physiological processes, including proliferation, migration, and differentiation. It thus is essential to investigate responses of cells corresponding to chemical and physical stimulations for cellular mechanics studies and other applications. Moreover, measurement of the stiffness of an interested cell is of great importance to comprehend the cellular response with respect to a specific biophysical or biochemical alteration. Hence, current study aims to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on nano-mechanical properties of cholesterol-manipulated cells. The NIH-3T3 fibroblastswere firstly incubated in cholesterol depletion solution. The cells were then insonified by a 1 MHz LIPUS for three minutes during the process of cholesterol restoration. In situ observation on the variations of living cells adhered on the glass substrate was monitored continuously. The cellular morphological changes were recorded for 8 hours. The nano-mechanical properties of cells were detected with nano-indentation by measuring the cellular membrane of a single cell. The cell properties seemed to be unchanged when adding with serum. On the other hand, the cholesterol depletion tended to lead a majority of cells to retract and become rounding corresponding to the increase of incubation time. The complications may be alleviated with the insonification of LIPUS. The elastic properties of those cholesterol-manipulated cells following LIPUS insonification tended to be improved. This study demonstrates that the variations of nano-mechanical property of cell membrane during cholesterol restoration and LIPUS insonification may be sensitively detected using the depth-sensing nano-indentation technique. The LIPUS insonification may be beneficial to the restoration of elastic properties of cholesterol-manipulated cells.