When vertebrates face acute stressors, their bodies rapidly undergo a repertoire of physiological and behavioral adaptations, which is termed the stress response. Rapid changes in heart rate and blood glucose levels occur via the interaction of glucocorticoids and their cognate receptors following hypothalamic-pituitary-adrenal axis activation. These physiological changes are observed within minutes of encountering a stressor and the rapid time domain rules out genomic responses that require gene expression changes. Although behavioral changes corresponding to physiological changes are commonly observed, it is not clearly understood to what extent hypothalamic-pituitary-adrenal axis activation dictates adaptive behavior. We hypothesized that rapid locomotor response to acute stressors in zebrafish requires hypothalamic-pituitary-interrenal axis activation. In teleost fish, interrenal cells are functionally homologous to the adrenocortical layer. We derived 8 frameshift mutants in genes involved in hypothalamic-pituitary-interrenal axis function: two mutants in exon 2 of mc2r (adrenocorticotropic hormone receptor), five in exon 2 or 5 of nr3c1 (glucocorticoid receptor), and two in exon 2 of nr3c2 (mineralocorticoid receptor). Exposing larval zebrafish to mild environmental stressors, acute changes in salinity or light illumination, results in a rapid locomotor response. We show that this locomotor response requires a functioning hypothalamic-pituitary-interrenal axis via the action of mc2r and the canonical glucocorticoid receptor encoded by nr3c1 gene, but not mineralocorticoid receptor (nr3c2). Our rapid behavioral assay paradigm based on hypothalamic-pituitary-interrenal axis biology can be used to screen for genetic and environmental modifiers of the hypothalamic-pituitary-adrenal axis and to investigate the effects of corticosteroids and their cognate receptor interactions on behavior.