In this paper, an integrated approach to study cellular hypoxic response under cyclic oxygen gradients is developed. In the experiments, a cell culture system based on a microfluidic device is constructed to generate cyclic oxygen gradients with fast response. The oxygen gradient is generated by alternatively introducing gases with specific compositions by a computer-controlled gas supply system into the microfluidic channels next to the cell culture one separated by thin channel walls. Observation of cellular hypoxic responses is performed using real-time fluorescence imaging of dyes sensitive to extra-and intra-cellular oxygen tensions as well as intracellular calcium concentrations. The capability of consistent cyclic oxygen gradient generation is confirmed by characterizing spatiotemporal oxygen tension profiles within the device. In the cell experiments, cellular hypoxic responses of human aortic smooth muscle cells (AoSMCs) and lung carcinoma epithelium (A549) cells including intracellular oxygen and calcium levels are measured. The relationships between the oxygen probe intensity and extracellular oxygen tension for both cell types are highly linear, and difference between the intensity measured from the two cell types is observed between 4 to 8% O2 culture environment. In addition, both cell types show detectable intracellular calcium concentration variation when the environmental O2 varies between normoxia and lower than 4%. The AoSMCs and A549 cells show decrease and increase of the intracellular calcium concentration during hypoxic response when facing change from normoxia to hypoxia environments, respectively. With the capability of real-time cellular responses monitoring under cyclic oxygen gradients, the developed approach provides a useful scheme to investigate cellular hypoxic response in vitro under microenvironments mimicking various in vivo physiological and pathological conditions.