The effect of the iron nitride phases, ε-Fe2-3N and γ′-Fe4N, on the fatigue strength was investigated. Pure iron was used to observe only the effect of nitride, excluding the effects of factors, such as residual stress, depending on the alloy composition and microstructural change according to working on the fatigue strength. In this work, ε and γ′ phases were respectively grown at a time on the surface of the pure iron specimens using the appropriate nitriding potential KN, the mixture rates of ammonia and hydrogen gases, at same temperature of 570 °C according to the Fe-N Lehrer diagram. Another γ′ phase was prepared by first growing the ε phase and then transformed from ε phase into γ′ phase by changing the KN at the same temperature of 570 °C in the 2-stage gas nitriding. The fatigue strengths of the iron nitride consisted of ε and γ′ phases, γ′ phase, and γ′ phase grown by the 2-stage gas nitriding were evaluated, respectively. As a result, first, it can be seen that the diffusion layer of ε phase was deeper than γ′ phase, but fatigue strength was lower. On the other hand, fatigue strength of both the γ′ phases are higher than that of the ε, and the fatigue strength of γ′ phase nitride grown by 2-stage gas nitriding was almost similar to that of γ′ phase nitride grown at a time, i.e., fatigue strength was not significantly related to diffusion depth and depended on nitride phases in this study. Secondly, we cannot clearly conclude that there was the difference in fatigue strength according to the thickness of nitride layer consisted of γ′ phase. However, it is clear that when ε phase was transformed to γ′ phase, fatigue strength had the same level as γ′ phase formed at one time.