Intracellular calcium homeostasis in adipocytes is important for the regulation of several functions and is involved in pathological changes in obesity and other associated diseases. Transient Receptor Potential Vanilloid 4 (TRPV4) channels are an important route for calcium entry that operates in a variety of cells and intervenes in a number of functions. In this study, the expression and operation of TRPV4 channels in human cultured adipocytes was evaluated using RT‐PCR, Western blotting, the whole‐cell patch‐clamp technique and fluorescence measurements to characterize these channels and determine intracellular calcium responses. Both the hypoosmolarity and 4alpha‐phorbol‐didecanoate (4αPDD), a specific TRPV4 agonist, induced a similar HC‐067047‐sensitive current, which was predominantly inward, and an intracellular Ca2+ concentration increase, which was exclusively dependent on extracellular calcium, and membrane depolarization. The current had a reverse potential of +31 ± 6 mV and exhibited preferential permeability to Ca2+. Insulin, which regulates metabolic homeostasis in adipocytes, attenuated the TRPV4‐mediated effects. These results confirm the function of TRPV4 in human cultured adipocytes and its regulation by insulin. J. Cell. Physiol. 9999: 1–10, 2015. © 2015 Wiley Periodicals, Inc.