Stressful manipulations can sensitize the behavior of an organism, increasing anxiety-like behavior after a delay; this long-term stress sensitization can represent the pathophysiological basis of trauma- and stress-related disorders (TRSDs), of which the most prevalent is post-traumatic stress disorder (PTSD). A role for the glutamate–nitric oxide pathway in this sensitization is implied by behavioral, neurophysiological and genomic data on different species. Here, we report on the long-term sensitization of anxiety-like behavior in zebrafish and the possible participation of nitric oxide in this process. Zebrafish exposed to a conspecific alarm substance (AS) show increased anxiety-like behavior at least 24 h after stimulus delivery. Blocking nitric oxide synthesis with l-NAME (5 mg/kg) 30 min, but not 90 min, after AS exposure blocks the sensitization of scototaxis and risk assessment, while treatment 90 min after exposure blocks the sensitization of thigmotaxis and erratic swimming; l-NAME was not effective when administered 30 min before AS exposure. These data suggest a participation of nitric oxide in the consolidation, but not in the initiation, of behavioral sensitization after predator threat.