Two-pore domain K(+) channels (K2P) mediate background K(+) conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K(+) channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH(o)) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH(o) affects these acid-sensitive K2P channels is not well understood. Here we show that in Na(+)-based bath solutions with physiological K(+) gradients, lowered pH(o) largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K(+) channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na(+) to K(+) relative permeability. Low pH(o)-induced inhibitions in these acid-sensitive K2P channels are more profound in Na(+)-based bath solutions than in channel-impermeable N-methyl-D-glucamine-based bath solutions, consistent with increases in the Na(+) to K(+) relative permeability and decreases in electrochemical driving forces of outward K(+) currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K(+) channels change ion selectivity in response to lowered pH(o), provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.