Background: Copper (Cu) is a pervasive environmental pollutant with significant hepatotoxic effects in animals. The endoplasmic reticulum (ER) interacts closely with lysosomes to maintain intracellular homeostasis. However, the role and mechanism of ER-lysosome crosstalk in Cu-induced liver injury in ducks remains unclear. To investigate this, we established both an in vivo model of Cu-exposed ducks and an in vitro model of duck hepatocytes, and added baicalin (Ba) to further explore its protective effects. Results: The results of this study demonstrated that exposure to Cu resulted in vacuolar degeneration and oxidative stress in duck hepatocytes, while ultrastructural observations revealed ER swelling and an increased number of autophagic lysosomes. Furthermore, Cu exposure significantly upregulated mRNA and protein levels related to ER stress, autophagy, and lysosomal membrane factors. It also markedly increased ER-lysosomal co-localization. Further experiments showed that knockdown of LAPTM4B significantly attenuated Cu-induced ER autophagy and reduced ER-lysosomal co-localization in hepatocytes. Molecular docking and molecular dynamics simulations confirmed that LAPTM4B has a stable binding site to Ba; in vitro experiments demonstrated that Ba could effectively alleviate Cu-induced ER-lysosome crosstalk in duck hepatocytes and reduce hepatocyte injury by targeting LAPTM4B; additionally, in vivo experiments showed that Ba significantly inhibits Cu-induced liver injury in ducks. Conclusions: In summary, the present study demonstrates that Cu exposure disrupts ER-lysosomal crosstalk in duck liver, leading to ER-lysosomal damage and subsequent hepatocyte injury. In contrast, Ba alleviates this injury by selectively targeting LAPTM4B, ultimately attenuating Cu-induced hepatotoxicity. Graphical