Macleaya cordatais a dominant plant of mine tailings and a zinc (Zn) accumulator with high Zn tolerance. In this study,M. cordataseedlings cultured in Hoagland solution were treated with 200 μmol·L−1of Zn for 1 day or 7 days, and then, their leaves were taken for a comparative analysis of the transcriptomes and proteomes between the leaves of the control and Zn treatments. Differentially expressed genes included those that were iron (Fe)-deficiency-induced, such as vacuolar iron transporterVIT, ABC transporterABCI17and ferric reduction oxidaseFRO.Those genes were significantly upregulated by Zn and could be responsible for Zn transport in the leaves ofM. cordata. Differentially expressed proteins, such as chlorophyll a/b-binding proteins, ATP-dependent protease, and vacuolar-type ATPase located on the tonoplast, were significantly upregulated by Zn and, thus, could be important in chlorophyll biosynthesis and cytoplasm pH stabilization. Moreover, the changes in Zn accumulation, the production of hydrogen peroxide, and the numbers of mesophyll cells in the leaves ofM. cordatawere consistent with the expression of the genes and proteins. Thus, the proteins involved in the homeostasis of Zn and Fe are hypothesized to be the keys to the tolerance and accumulation of Zn inM. cordata. Such mechanisms inM. cordatacan suggest novel approaches to genetically engineering and biofortifying crops.