The effects of nitric oxide (NO) donor sodium nitroprusside (SNP) and various growth regulators on callus induction and shoot organogenesis in the commercially and pharmaceutically important medicinal plant Valeriana jatamansi is presented. Four different growth regulators at different concentrations were tested for their callusing efficiency followed by supplementation of SNP to the best responding growth regulator. Maximum callusing frequency (91.18%) was observed with the supplementation of 1.5 mg/L 1-Naphthaleneacetic acid + 15 μM SNP. 10% Coconut water (CW) was found to be the best growth regulator for the induction of shoots. The multiple shoot formation was also found to be enhanced with the supplementation of SNP. Maximum response for in vitro shoot multiplication was observed when the medium was supplemented with 10% CW + 15 μM SNP (89.32%) as compared to shoots induced in medium supplemented with 1.5 mg/L Benzyl aminopurine + 15 μM SNP (79.46%) respectively. No separate root induction medium was required as CW and CW + SNP supplementation was found to be sufficient enough to produce profuse rooting of the in vitro regenerated plants. Results revealed that SNP enhanced the callus and shoot induction frequencies. Supplementation of SNP also significantly reduced callus browing, allowed the tissues to recover and regenerate. This proposed in vitro culture system will be an effective mass propagation strategy for both commercial utilization and conservation of this highly valued plant species. First report using SNP and growth hormone supplementation for enhanced propagation in Valeriana jatamansi presented. Supplementation of SNP enhanced the callus, multiple shoot induction and reduced callus browning in the in vitro cultures of V. jatamansi.