Globally, crop production has been widely threatened by contamination of arable lands with heavy metals including Nickel (Ni). Stress-relief molecule melatonin (ME) has been widely used to mitigate the phytotoxicity induced by heavy metals. The current study aimed to explore the response to Ni stress and the alleviating role of ME in boosting Ni-stress tolerance in tomato seedlings. The roots of tomato seedlings pretreated with ME (100 mu M) for 3 days, followed by applied Ni (50 mu M) for 7 days. The treatments were composed of (1) control (CK); (2) melatonin (ME, 100 mu M); (3) nickel (Ni, 50 mu M); and (4) melatonin and nickel treatment (ME+Ni, 100 mu M + 50 mu M). Nickel toxicity noticeably inhibited plant growth and biomass production by impairing the root architecture, photosynthesis process, nutrient uptake, and antioxidant enzymes. Conversely, ME-supplementation inhibited Ni-induced growth damage, improved root architecture, nutrient uptake, pigment contents, and leaf gas exchange parameters, and decreased Ni-accumulation. Furthermore, the electrolyte leakage (EL), malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation were significantly reduced in ME-treated seedlings via improving antioxidant enzyme activity as well as upregulation of their encoding gene expression. In conclusion, our findings provide a shred of substantial evidence that ME improved Ni-induced phytotoxicity in tomato seedlings, mainly by improving the root architecture, biomass production, mineral homeostasis (reducing nickel accumulation in plants), and photosynthetic efficiency.