It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning ∼1 mm2 with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.