Key message <p>An endoplasmic reticulum-localized Cu transporter, PhHMA5II1, interacts with copper chaperones and plays an important role in Cu detoxification in petunia.</p> Abstract <p>Copper (Cu) is an essential element for plant growth but toxic when present in excess. In this study we present the functional characterization of a petunia (<i>Petunia hybrida</i>) P_1B-type heavy-metal ATPases (HMAs), PhHMA5II1. Heterologous expression of PhHMA5II1 in yeast (<i>Saccharomyces cerevisiae</i>) showed Cu transport activity. The expression of <i>PhHMA5II1</i> in roots and shoots was unaffected by excess Cu. CRISPR/Cas9-edited mutant lines and PhHMA5II1 overexpressing transgenic plants were generated to investigate the functions of PhHMA5II1 in petunia. The PhHMA5II1 knockout mutant was hypersensitive to excess Cu and accumulated more Cu in roots compared to wild-type petunia. Overexpression of PhHMA5II1 enhanced Cu tolerance and reduced Cu accumulation in roots. Furthermore, PhHMA5II1 localized in endoplasmic reticulum, and the localization was unaffected by excess Cu. Yeast two-hybrid experiments and bimolecular fluorescence complementation assays demonstrate that PhHMA5II1 interact with petunia copper chaperons, PhATX1 and PhCCH. Finally, RNA-sequencing revealed that knockout <i>PhHMA5II1</i> affected the expression of genes involved in cell-wall organization, copper ion homeostasis, and response to oxidative stress. Taken together, PhHMA5II1 plays an important role in Cu detoxification in petunia.</p>