Membrane transport proteins, which include transporters and channels, are delicate protein machineries that mediate the exchange of a variety of substances across biomembranes. Accumulated structural and functional knowledge allows for the de novo design of transport proteins with new structures that do not exist in nature. Analysis based on these novel proteins provides new insights into the principles that govern protein assembly, conformational change, and substrate recognition. Here, we review the advances in the de novo design of transporters and channels over recent years and highlight the challenges and opportunities in this field.