Investigations have been conducted on the fatigue behavior of Ti-6Al-4V alloy with the bimodal microstructure in air at room temperature, in vacuum at room temperature and in vacuum at low temperature (100K), respectively. The results show that the fatigue life of this alloy is longer in vacuum and/or at low temperature than that in air at room temperature. The combination of vacuum and low temperature can much improve the fatigue properties of this alloy because of their purifying effect on fatigue cracks propagation. SEM observation of fatigue fracture surface indicates that all the fatigue cracks initiate in the sample surface when Nf ≦106 cycles. The above three testing conditions lead to different modes of fatigue crack propagation and therefore much different morphology of fatigue fracture.