The aim of this study was to investigate the crosstalk between oxidative stress and autophagy through the ROS/HO-1/NQO1 pathway caused by Cu. Duck renal tubular epithelial cells were treated in Cu sulfate (CuSO₄) (0, 100 and 200 μM) for 12 h, and in the combination of CuSO₄ (200 μM) and reactive oxygen species (ROS) scavenger (butyl hydroxyanisole, BHA, 100 μM), or HO-1 inhibitor (zinc protoporphyrin, ZnPP, 10 μM) for 12 h. Results revealed that Cu could significantly elevate the levels of intracellular ROS, superoxide dismutase, hydrogen peroxide, malondialdehyde, glutathione, simultaneously reduce catalase and glutathione peroxidase levels, and upregulate HO-1, SOD-1, CAT, NQO1, GCLM mRNA levels and HO-1, SOD-1 protein levels. Additionally, Cu could observably increase the number of autophagosomes, acidic vesicle organelles (AVOs) and LC3 puncta; upregulate mRNA levels of mTOR, Beclin-1, ATG7, ATG5, ATG3, LC3II and protein levels of Beclin-1, LC3II/LC3I, downregulate LC3I mRNA level. Both treatments with BHA and ZnPP could significantly alleviate the changes of antioxidant indexes levels and ROS accumulation, reduce the increase of the number of autophagosomes, AVOs and LC3 puncta, and mitigate the above changed oxidative stress and autophagy related mRNA and protein levels induced by Cu. In summary, our findings indicated that excessive Cu could induce oxidative stress and autophagy by activating the ROS/HO-1/NQO1 pathway, and inhibition of HO-1 might attenuate Cu-induced oxidative stress and autophagy in duck renal tubular epithelial cells.