The objective of this work was to develop and characterize a poly(dimethylsiloxane) device with an integrated active cooling function able to carry out capillary electrophoresis separations. Polymer-based microdevices are indispensable to recent advances in biomedical analysis. In particular, they have been applied to many microfluidic platforms owing to their low cost, ease of fabrication, and versatility in preparing complex microstructures. However, when applied to capillary electrophoresis separations, polymer microfluidic structures present an inherent disadvantage compared to glass and Si structures; they have a lower thermal conductivity than glass and Si. Although miniaturized devices allow operation at high electric fields, they face separation efficiency limitations due to Joule heating. There is, therefore, a strong need of developing capillary electrophoresis microfluidic structures with active cooling in order to operate at a higher electric field and potentially increase separation efficiency in these microdevices. A poly(dimethylsiloxane)/glass hybrid microfluidic capillary electrophoresis system is presented, where Joule heating was minimized by using an integrated active cooling function. Two poly(dimethylsiloxane) slabs with embedded microfluidic structures were irreversibly sealed on both sides of a thin glass slide. The top poly(dimethylsiloxane) slab was used to carry out capillary electrophoresis separations, whereas the bottom poly(dimethylsiloxane) slab was employed to cool down the buffer solution used during the capillary electrophoresis separation. As demonstrated on current versus voltage plots and on capillary electrophoresis electropherograms, capillary electrophoresis separation was able to be operated at a higher electric field when using the cooling function. The cooling rate was adjustable by varying the flow rate and the initial temperature of the liquid flowing in the cooling microfluidic structure.