Polyurethane (PU) membranes usually have high gas permeability but low selectivity. It was supposed that an increase of the hard-segment content in PU membranes would enhance the selectivity since the hard segment has a higher oxygen affinity and could reduce the chain mobility. However, an attempt to increase the O-2/N-2 selectivity by increasing the hard-segment content failed. It was found that increase of the hard-segment content did increase the gas solubility but the solubility ratio of oxygen to nitrogen was not increased. Increase of the hard-segment content also reduced the permeabilities of both oxygen and nitrogen, but no improvement of the O-2/N-2 diffusivity ratio was observed. The microstructure of polybutadiene-containing PU was examined by DSC and FTIR measurements. It was found that hydrogen bonding between hard segments was the cause of the failure of selectivity improvement. Avoiding the formation of hydrogen bonds between the hard segments is the key for a successful improvement of the O-2/N-2 selectivity of the PU membranes.