<p>Chromium (Cr) is one of the most pervasive hazardous trace elements in agricultural soils due to anthropogenic activities. However, the uptake mechanism and subcellular distribution of Cr in medicinal plants are limited. Therefore, this study investigated the uptake and subcellular distribution of Cr in two accessions of <i>Andrographis paniculata</i> (AP1 and AP3). Results demonstrated that AP1 had a higher tolerance index and lower accumulation of Cr than AP3. Higher accumulation of Cr in the root and lower upward translocation were observed in AP1 compared to AP3. The sub-cellular distribution of Cr demonstrated more sequestration of Cr in the cell wall fraction of AP1 and the soluble fraction of AP3. The Michaelis-Menten kinetics displayed low Km (high affinity) and high Vmax/Km ratio in AP3 compared to AP1. The lower concentration and proportion of Cr in the organelle fraction of root and aboveground tissue of AP1 (the least Vmax/Km ratio) could be the reason for the minimum disruption of normal metabolism and consequently responsible for tolerance among the accessions. The Ca, Zn, Fe, and S were the major ions influencing the Cr uptake and accumulation in two accessions. The study provides the mechanism for the differential uptake of Cr in two genotypes, which can be helpful in their safe cultivation.</p>