The dose rate effect significantly impacts accelerated assessment experiments of the radiation resistance of critical structural materials in nuclear reactors. This study investigated the role of the dose rate on the irradiation hardening and defect cluster formation in a low-Cu reactor pressure vessel (RPV) steel A508-3 under 3.5 MeV Fe13+ ions irradiation at 290 °C, with chosen dose rates of 2.8 × 10–5, 1.4 × 10–4 and 2.8 × 10–4 dpa/s. Nano-indentation and atom probe tomography (APT) techniques were employed to characterize the mechanical property changes and defect clustering behavior, respectively. Nanoindentation results revealed that for a fixed dose, greater hardening was observed in the sample irradiated at a lower dose rate, and conversely, less hardening at a higher dose rate. APT results for samples with 3.0 dpa demonstrated that defect clusters exhibited characteristics of small size and low density under irradiation at a dose rate of 2.8 × 10–4 dpa/s, and large size and high density at a dose rate of 2.8 × 10–5 dpa/s. The ratio of solutes to Fe elements in a cluster also varied with changes in dose rate. The change in hardening, cluster size, number density and cluster composition suggested that the evolution of defect clusters was primarily driven by ballistic mixing, rather than radiation-enhanced diffusion, under heavy ions irradiation within the dose rate range of 2.8 × 10–5–2.8 × 10–4 dpa/s.