Objectives: A traditional herb Commiphora wightii (Arnott.) is well known for the management of different ailments, including hypolipidemic and hypocholesterolemic disorders. Overexploitation has resulted in enlisting it under endangered species list; thus, in vitro studies are desirable to circumvent further depletion of the wild population. The objective of our study is to analyze the effect of salinity stress on growth and guggulsterone content in callus cultures. Materials and Methods: The establishment of leaf-derived calli of C. wightii and the effect of salinity stress such as calcium chloride (CaCl2), sodium chloride (NaCl), potassium chloride, and ammonium sulfate at different concentrations and time periods on callus growth and steroid biosynthesis were studied. High-performance liquid chromatography and liquid chromatography-mass spectrometry/mass spectrometry-based spectrometric systems were carried to identify the guggulsterones. Results: The 50 mg/L CaCl2-induced callus was found to have the highest content of guggulsterones after 15 months of incubation as compared to 9 and 12 months and indicated a positive correlation between callus age and guggulsterone biosynthesis. Conclusion: Salinity stress played an important role in callus growth of C. wightii, while calcium-induced calli showed in vitro synthesis of guggulsterones, and this can be further explored for the synthesis of guggulsterones in vitro on large commercial scale using bioreactor, and thus helps to conserve the endangered species in its natural habitat.