Cadmium (Cd) is very toxic to plant cells and Cd²⁺ stress induces programmed cell death (PCD) in Nicotiana tabacum L. cv. bright yellow-2 (BY-2) cells. In plants, PCD can be regulated through the endoplasmic reticulum (ER) stress–cell death signaling pathway. However, the mechanism of Cd²⁺-induced PCD remains unclear. In this study, we found that Cd²⁺ treatment induced ER stress in tobacco BY-2 cells. The expression of two ER stress markers NtBLP4 and NtPDI and an unfolded protein response related transcription factor NtbZIP60 were upregulated with Cd²⁺ stress. Meanwhile, the PCD triggered by prolonged Cd²⁺ stress could be relieved by two ER chemical chaperones, 4-phenylbutyric acid and tauroursodeoxycholic acid. These results demonstrate that the ER stress–cell death signaling pathway participates in the mediation of Cd²⁺-induced PCD. Furthermore, the ER chaperone AtBiP2 protein alleviated Cd²⁺-induced ER stress and PCD in BY-2 cells based on the fact that heterologous expression of AtBiP2 in tobacco BY-2 cells reduced the expression of NtBLP4 and a PCD-related gene NtHsr203J under Cd²⁺ stress conditions. In summary, these results suggest that the ER stress–cell death signaling pathway regulates Cd²⁺-induced PCD in tobacco BY-2 cells, and that the AtBiP2 protein act as a negative regulator in this process.