The effects of the deactivation of nanoscale zero-valent iron (NZVI), induced by humic acid (HA) and by the retention of NZVI in water, on nitrate reduction were investigated using a kinetic study. Both the nitrate removal and generation of ammonia were significantly inhibited as the HA adsorption amount and retention time were increased. However, HA removal was greatly enhanced when the NZVI was used after 1 d or 25 d of retention in water. The results are caused by the formation of iron oxides/hydroxides, which increased the specific surface area and the degree of NZVI aggregation which was observed by transmission electron microscopy (TEM). However, the nitrate reduction was greater at the beginning of reaction in the presence of HA when fresh NZVI was used, because of the enhanced electron transfer by the HA in bulk phase and on NZVI surface as train sequences. The pseudo second order adsorption kinetic equation incorporating deactivation and a Langmuir-Hinshelwood (LH) type kinetic equation provided accurate descriptions of the nitrate removal and ammonia generation, respectively. The deactivation constant and the reaction rate constant of the LH type kinetic equation were strongly correlated with the HA amount accumulated on NZVI. These results suggest that the HA accumulation on the NZVI surface reactive sites plays the dominant role in the inhibition and the inhibition can be described successfully using the deactivation model. The HA accumulation on NZVI was verified using TEM.