Madder (Rubia tinctorum L.) is known as one of the most useful multipurpose plants. Madder root and rhizome contain alizarin. Pharmacological studies have shown that alizarin has a certain degree of selective inhibitory effects on cancer cells and it is used as a targeted drug for cancer treatment. Native knowledge of local farmers about the madder is that its roots grow better in calcareous soils, and its content of alizarin will be higher. Therefore, in this paper, the effects of different ratios of CaCO 3 (0, 20, 50, 100, and 150 kg ha -1 ) and triple-superphosphate fertilizer (0, 50, 100, and 150 kg ha -1 on physiological traits of madder were studied. Results revealed that certain physiological traits (total fresh weight, root fresh weight, root length, net assimilation rate, relative growth rate, and root relative growth rate) were increased under mediumlevel (50 kg ha -1 ) CaCO 3 and triple superphosphate treatments, but higher concentrations decreased the mentioned growth parameters, because the high concentrations of calcium carbonate caused the increase in concentrations, it negatively affected the results because they are complex compounds that cannot be used. These results suggest that low rates of CaCO 3 to highlight incremental stimulations of total fresh weight (79.42%) and alizarin content (26.72%) in madder roots and stimulate the interest for further studies and exploitation in the pharmaceutical industry.