Acute tryptophan depletion (ATD) is extensively used to investigate the role of central serotonin (5-HT). However, several studies reported that ATD had no significant effect on central 5-HT concentration and some ATD-induced changes was independent of 5-HT in the rodent brain. Therefore, the potential mechanism of ATD might not be ascribed solely to changes in the central 5-HT system. In recent studies, evidence suggests that nitric oxide synthase (NOS) is closely associated with ATD-induced changes in modulation of cerebral blood flow and metabolism, cognitive, and locomotor activity. Thus, NOS is implicated to be an underlying factor contributing to ATD-induced changes. In the present study, the effect of ATD upon central NOS levels in the rat was evaluated. Male Sprague-Dawley (SD) rats were orally administered a tryptophan-free protein-carbohydrate mixture. Then, ATD effects upon affective behavior and spatial memory were assessed by the forced swimming test (FST) and Morris water maze test, respectively. Further, NOS activity and neuronal NOS (nNOS) protein levels in the hippocampus were measured after ATD. Our experimental results showed that ATD had no influence on affective behavior in the FST or spatial memory in SD rats. Interestingly, a significant reduction of both constitutive NOS activity and nNOS protein levels after ATD was found in the hippocampus. These findings demonstrate ATD does not influence affective behavior and spatial memory despite a direct effect on hippocampal NOS. Our study might provide a valuable clue for exploring earlier reported ATD-induced behavioral and neurochemical changes in rodents.