Pd and PdCu alloy membranes are promising candidates for separating hydrogen from mixed gas streams due in part to their infinite selectivity to hydrogen separation. However, other gases such as CO can inhibit hydrogen transport across Pd-based membranes. In this work, the mechanism by which CO inhibits hydrogen transport across a 25 mu m-thick Pd47Cu53 (mol%) membrane is investigated by operando infrared-reflection absorption spectroscopy (IRAS) in the 373-533 K temperature range. In the absence of hydrogen, CO adsorbs on three different sites on the PdCu surface: (1) bridging between contiguous Pd atoms, (2) on top of isolated Pd atoms surrounded by Cu atoms, and (3) on top of oxidized Cu atoms. CO induces agglomeration of isolated Pd atoms on the PdCu surface, which is driven by the higher stability of CO adsorbed on bridging sites between Pd atoms than on isolated Pd atoms surrounded by Cu atoms. The rate of hydrogen permeation across the PdCu alloy membrane decreases with increasing CO concentration in the feed gas, and the poisoning effect of CO is more severe at lower temperatures. CO inhibits hydrogen transport across the membrane by adsorbing only on Pd sites on the PdCu surface and blocking H-2 dissociation on these sites. Due to the weaker interaction of CO with PdCu alloy surfaces than with Pd, the PdCu alloy is more resistant to CO poisoning than pure Pd.