The lack of information on how biological systems respond to low-dose and low dose-rate exposures makes it difficult to accurately assess the carcinogenic risks. This is of critical importance to space radiation, which remains a serious concern for long-term manned space exploration. In this study, the γ-H2AX foci assay was used to follow DNA double-strand break (DSB) induction and repair following exposure to neutron irradiation, which is produced as secondary radiation in the space environment. Human lymphocytes were exposed to high dose-rate (HDR: 0.400 Gy/min) and low dose-rate (LDR: 0.015 Gy/min) p(66)/Be(40) neutrons. DNA DSB induction was investigated 30 min post exposure to neutron doses ranging from 0.125 to 2 Gy. Repair kinetics was studied at different time points after a 1 Gy neutron dose. Our results indicated that γ-H2AX foci formation was 40% higher at HDR exposure compared to LDR exposure. The maximum γ-H2AX foci levels decreased gradually to 1.65 ± 0.64 foci/cell (LDR) and 1.29 ± 0.45 (HDR) at 24 h postirradiation, remaining significantly higher than background levels. This illustrates a significant effect of dose rate on neutron-induced DNA damage. While no significant difference was observed in residual DNA damage after 24 h, the DSB repair half-life of LDR exposure was slower than that of HDR exposure. The results give a first indication that the dose rate should be taken into account for cancer risk estimations related to neutrons.