In this paper, the effects of hydrogen content and temperature on the fatigue crack initiation and propagation behavior of Zircaloy-4 are investigated by in-situ scanning electron microscope observation. The results show that, at room temperature, with the increase of hydrogen content, the tensile strength increases and the elongation decreases, but the fatigue lifetime (fatigue crack initiation lifetime and fatigue failure lifetime) first increases and then decreases. The cut-off point is at about 200 ppm hydrogen content. The fatigue crack growth rate increases with the increase of hydrogen content. At 300 °C, with the increase of the hydrogen content, tensile strength, elongation and fatigue lifetime of Zircaloy-4 alloys exhibit the same variations as those at room temperature. However, the fatigue crack growth rate is less affected by the hydrogen content. The results also show that, with the same hydrogen content, the fatigue lifetime at 300 °C is longer than that at room temperature, and the fatigue crack growth threshold at 300 °C is lower than that at room temperature. The fatigue crack propagation path is strongly affected by the hydride and grain boundaries. The hydrogen content has a significant effect on the fatigue crack initiation mechanism. For the un-hydrided sample, the fatigue cracks initiate at the sub-surface. With the increase of hydrogen content, the fatigue cracks tend to initiate at the surface of the sample.