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This paper presents the first experimental study to realize a DNA separation chip based on the self-focusing effect in a micropillar array. The present self-focusing chip redistributes DNA molecules within a specific area of micropillar arrays based on the size- and field-dependent nonlinearity of DNA drift velocity. Compared to conventional electrophoresis chips, the present self-focusing chip reduces a substantial amount of the separation channel length, the influence of sample starting location, and the necessity of time-consuming continuous monitoring process. We focus on the design of DNA self-focusing chips, with identifying the nonlinearity of DNA drift velocity using three different DNA molecules including λ DNA (48.5 kbp), micrococcus DNA (115 kbp), and T4 DNA (169.8 kbp) in microfabricated test chips. It is demonstrated that the present DNA self-focusing chips have potentials not only for the miniaturization of DNA analysis systems, but also for the tunable capability of the target DNA size to be separated, trapped and extracted.