Physiological depletion of tryptophan, the precursor to serotonin has been shown to alter mood and cognition in both humans and rodents. Few studies have investigated the neurochemical and behavioural effects associated with tryptophan depletion in mice. Given that BALB/c and C57BL/6J mice differ in tryptophan hydroxylase (TPH) functionality, serotonin levels and behavioural phenotype, we hypothesised that a differential strain response to chronic dietary tryptophan manipulations would be observed. Therefore, the effects of four chronic dietary tryptophan manipulations were investigated, the diets include a depleted diet (0% tryptophan, TRP−), a deficient diet (0.25% tryptophan, TRP−/+), an enhanced diet (1.25% tryptophan, TRP+) and a control diet (0.7%). Diet-induced alterations in peripheral and central tryptophan levels and brain serotonin turnover were determined by high performance liquid chromatography. In addition, dietary-induced alterations in behaviour were assessed in several commonly used tasks. Peripheral and central tryptophan levels and consequently central serotonergic turnover were significantly decreased by the TRP− diet in both strains, however, no effect of tryptophan supplementation was observed on tryptophan or serotonin levels. Dietary tryptophan manipulation induced pronounced behavioural effects, particularly in nesting behaviour where a reduction in nesting was observed following depletion and an increase in nesting behaviour was observed with enhanced tryptophan in both strains. Additionally, depletion produces an anxiolytic-like effect and did not impede locomotion. This study demonstrates significant alterations in the levels of tryptophan, serotonin turnover and behaviour following chronic dietary tryptophan depletion.