In this paper, the effect of microstructure on the room-temperature hydrogen absorption behavior of Ti-6Al-4V alloy was studied through electrolytic hydrogen charging method. The results show that Ti-6Al-4V alloys with different microstructures show obviously different room-temperature hydrogen absorbing ability. The hydrogen absorbing ability of the alloy with duplex microstructure is the weakest and that of the alloy with widmanstatten microstructure is the strongest at room temperature. The influence of the microstructure characteristics of β phase is stronger than that of α phase on the capability for room-temperature hydrogen absorption. The Ti-6Al-4V titanium alloy with duplex microstructure should be the one with a perfect resistance to the hydrogen embrittlement.