Objective <p>To explore the inhibitory effects of <i>Nardostachys Jatamansi DC.</i> volatile oil (NJVO) on psychological factors substance P (SP)/cortisol (CORT)-induced hyperpigmentation.</p> Methods <p>The model of psychologically-induced hyperpigmentation of B16F10 cells was created using SP (10 nmol/L) + CORT (10 µmol/L) for 72 h. The levels of melanin content, tyrosinase (TYR) activity using NaOH lysis and L-dihydroxyphenylalanine (L-DOPA) oxidation methods were assessed, respectively. The effect of NJVO on SP/CORT-induced normal human skin tissue pigmentation was detected by Masson staining. Protein expressions of tyrosinase-related protein 1 (TRP-1), tyrosinase-relative protein 2 (DCT), and microphthalmia-associated transcription factor were determined using Western blot. The melanosome number, maturation, and melanosomal structure changes were detected through transmission electron microscopy and immunofluorescence experiments. <i>In vivo</i>, zebrafish pigment content was evaluated in SP/CORT-induced zebrafish hyperpigmentation model.</p> Results <p>NJVO significantly reduced the melanin content (<i>P</i>&lt;0.01) and inhibited tyrosinase activity (<i>P</i>&lt;0.01), the pigmentation of the normal skin tissue in the NJVO group was significantly lower than that in the SP/CORT group (<i>P</i>&lt;0.05). And NJVO considerably downregulated expressions of melanogenesis-related proteins (TYR, TRP-1, DCT) in cells (<i>P</i>&lt;0.01). In addition, the number of melanosomes was decreased and the dentrites formation of B16F10 cells was inhibited after NJVO treatment (<i>P</i>&lt;0.01). <i>In vivo</i>, NJVO significantly reduced the pigment content in the zebrafish body (<i>P</i>&lt;0.01).</p> Conclusion <p>NJVO effectively reversed SP/CORT-induced hyperpigmentation by suppressing the activity and expression of TYR and TRPs and inhibiting melanosome maturation in mouse B16F10 melanoma cells.</p>