Cadmium (Cd) poses a threat to food safety and human health. Plant defensins are small peptides that involved in Cd metabolism. however, the function of tobacco phyto-defensin protein NtCAL1 in Cd metabolism remains poorly understood. Here we identified the effects of NtCAL1 from tobacco on Cd metabolism. We provide evidence that NtCAL1 is located in the cell wall and varied expressed in organs of developmental stages. The overexpression of NtCAL1 has been demonstrated to enhance the content of AsA and antioxidant enzyme (CAT and APX) activities, as well as to positively regulate the expression of NtRAMP3, NtHMAα and NtHMAβ. The NtCAL1 was identified as a positive regulator of cadmium (Cd) translocation from the root to the shoot. Furthermore, the accumulation of Cd was inhibited by Cd foliar inhibitors. In conclusion, we propose that NtCAL1 acts synergistically with other proteins involved in Cd uptake and transport, thereby enhancing Cd tolerance through the upregulation of antioxidant substances and the activation of antioxidant enzymes.The NtCAL1 provides a significant potential tool for the development of low-Cd tobacco, while remediation of Cd contaminated field soils.