Hyoscyamus niger L. is a plant with high medicinal value in terms of its tropane alkaloids. In this study, the effects of cadmium chloride (CdCl2) application on root growth and secondary metabolite production in adventitious root cultures of H. niger were investigated. For this purpose, CdCl2 was applied to the roots at different concentrations (1, 2, and 3 mM) for 24, 48, 72, and 96 h. After harvest, the roots were evaluated in terms of not only growth parameters including fresh root weight, dry root weight, and root growth index but also the production of tropane alkaloids and phenolic compounds. In the study where it was found that CdCl2 application had a negative effect on root growth, the highest root growth parameters were determined in control roots harvested after 96 h. The greatest value for hyoscyamine was determined in the roots harvested 24 h after 3 mM CdCl2 application while 2 mM CdCl2 applied for 24 h is the most appropriate application in terms of scopolamine production. On the other hand, the application of 3 mM CdCl2 for 72 h proved to be the most encouraging application to increase the production of phenolic compounds. Principal component analysis (PCA) transferred the parameters examined in this study to five principal components (PCs) with eigenvalues greater than 1, explaining approximately 78.07 % of the total variances in roots. In conclusion, the application of CdCl2 was found to have promising effects on the production of tropane alkaloids and phenolics depending on concentration and harvest time.