We report an on-chip platform for low-intensity pulsed ultrasound (LIPUS) stimulation of cells directly cultured on a biocompatible surface of a transparent ultrasound transducer (TUT) fabricated using lithium niobate. The high light transmittance (>80%) and compact size (3 mm × 3 mm × 2 mm) of TUTs allowed easy integration with powerful optical microscopy techniques with no additional acoustic coupling and risk for contamination. TUTs were excited with varying acoustic excitation parameters (voltage amplitude and duty cycle) and resulting live cell calcium signaling was simultaneously imaged using time-lapse confocal microscopy, while the temperature change was measured by a thermocouple. Quantitative single-cell fluorescence analysis revealed the dynamic calcium signaling responses and together with the temperature measurements elucidated the optimal stimulation parameters for non-thermal and thermal effects. The fluorescence change profile was distinct from the recorded temperature change (<1 degree Celsius) profile under LIPUS treatment conditions. Cell dead assay results confirmed cells remain viable after the LIPUS treatment. These results confirmed that the TUT platform enables controllable, safe, high-throughput, and uniform mechanical stimulation of all plated cells. The on-chip LIPUS stimulation using TUTs has the potential to attract several in vitro and in vivo biomedical applications such as controlling stem cell differentiation and proliferation, studying biomechanical properties of cancer cells, and gaining fundamental insights into mechanotransduction pathways when integrated with state-of-the-art high-speed and high-resolution microscopy techniques.