Abstract: In fusion reactors, high-energy neutrons continuously generate helium atoms through (n,α) transmutation reactions with structural materials, leading to degradation of material service performance and posing a serious threat to the safe operation of the reactors. Therefore, revealing the influence of temperature on helium-induced defect evolution and mechanical behavior in low-activation steels at the atomic scale is a key scientific foundation for designing high-performance fusion structural materials. This study employs molecular dynamics simulations to investigate the effect of temperature on defect evolution and mechanical behavior in single-crystal iron containing 5000 appm helium. The results show that point defects (Frenkel Pairs, FPs) rapidly increase to a peak before stabilizing, and higher temperatures promote an increase in the peak FP concentration. At T = 773 K, the peak FP content accounts for about 0.5% of the system. Interstitial helium atoms preferentially combine with vacancies to form stable He–V complexes, and the number of He–V complexes also increases with temperature. Regarding cluster defects, elevated temperature (T = 773 K) promotes an increase in the number of small clusters and the formation of large clusters (cluster size = 16), while vacancy clusters are less affected by temperature. The clustering rate of vacancy clusters is generally higher than that of interstitial clusters. After helium behavior stabilizes, the model is cooled to room temperature and subjected to uniaxial tensile testing. It is found that large interstitial clusters and He–V complexes jointly enhance the local elastic modulus while reducing the peak stress and peak strain. Plastic deformation is primarily governed by the dislocation glide mechanism. The defect structures formed during evolution at 300 K facilitate the activation of multiple slip systems, resulting in plastic deformation dominated by dislocation glide on multiple slip systems. With the increasing number of He–V complexes, the large defect clusters formed during evolution at 773 K exert a stronger hindering effect on dislocation glide, leading to dislocation activity that is mainly concentrated on a limited number of slip systems and exhibits a relatively discrete spatial distribution. This study reveals the regulatory role of temperature on defect formation, evolution, and mechanical properties in helium-implanted single-crystal iron, providing theoretical insights for developing iron-based fusion materials with superior irradiation resistance.