Among the toxic heavy metal(loid)s, cadmium (Cd) and arsenic (As) have devastating effects on crop productivity and human health. In plants, mechanisms of detoxification of Cd and As proceed via a glutathione (GSH) dependent common pathway, despite their different modes of toxicity. In this context, the present study aimed to investigate how the common detoxification mechanisms for Cd and As influence the physiological and biochemical responses of seedlings of an important plant used for phytoremediation purposes, Brassica juncea, under Cd and As stress. We demonstrated that Cd and As trigger mostly distinct, but partly overlapping, responses in B. juncea. Exposure of B. juncea seedlings to 100 μM Cd stress for 12 days in a hydroponic system led to a significant reduction in the growth of roots and shoots, and in total chlorophyll content. However, As stress caused a decline in root length only. High-performance liquid chromatography analyses revealed a significant increase in cysteine levels in roots and shoots in response to As stress compared with control and Cd-treated plants. Concomitant elevated sulfur content in response to As stress was observed in roots and shoots. In terms of GSH content, Cd and As triggered similar responses, with a significant decrease in GSH in roots, and non-significant changes in shoots, compared with untreated plants. Inductively coupled plasma-atomic emission spectroscopy revealed that under Cd stress, plants preferentially accumulated zinc (Zn) in the roots compared with iron (Fe) and manganese (Mn). Responses in roots under As and Cd stress were similar with respect to Fe accumulation but opposite in terms of Zn and Mn accumulation. Our data provide valuable insights for design of future strategies for sustainable plant growth on As and Cd polluted soils.