Heavy metal exposure impacts basic cellular processes and results in serious toxicological effects. Pb2+ can activate the response to endoplasmic reticulum (ER) stress by protein denaturation, changing intracellular calcium homeostasis, and inducing cell death. As an ER retention protein, 78-kDa glucose-regulated protein (GRP78) can relieve the Pb2+-induced ER stress and enhance cell viability. We previously showed that heavy metal ions such as Pb2+ etc. are harmful to fish cell lines in a time- and dose-dependent manner. The phenomenon is accompanied by the increasing accumulation of grass carp GRP78 (CiGRP78), which can protect the cells from heavy metal ion cytotoxicity. Here, we investigated the mechanism in which CiGRP78 exerted its protective function. Using metal ions affinity elution method and fluorescent spectral analysis, we showed that CiGRP78 could respectively form a complex with Calcium, Lead and Cadmium ions, especially with Lead ion in vitro. However, another ER retention protein CiGRP94 could not bind to Pb2+, highlighting the functional differentiation might exist in CiGRP78 and CiGRP94 in regulating heavy metal cytotoxicity. Our results suggested that CiGRP78 might increase cellular tolerance to Pb2+ via the direct interaction with it.