The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca²⁺]_i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca²⁺]_i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca²⁺]_i. Step changes in shear stress resulted in a rapid increase in [Ca²⁺]_i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca²⁺]_i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm² stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm² stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca²⁺]_i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.