Protoporphyrinogen oxidase (PPOX1) is a key enzyme in the pathway of chlorophyll biosynthesis, and it is used to explore the function of PPOX1 gene in apple. In this study, Malus halliana, as a apple rootstock was used as the test material. The MhPPOX1 gene fragment was cloned by PCR and bioinformatics analysis and functional identification were conducted. Using the method of agrobacterium mediated genetic transformation of Nicotiana tabacum L. and Arabidopsis thaliana, the function of MhPPOX1 under iron deficiency stress was further analyzed, and resistance analysis of transgenic N. tabacum L. and A. thaliana were conducted. The results show that: (1) the MhPPOX1 gene fragment of M. halliana was successfully cloned and identified as apples MhPPOX1 (serial number: loc103444480). The open reading frame of MhPPOX1 gene is 1 644 bp, encoding 547 amino acids, and the isoelectric point is 8.98. Phylogenetic tree analysis showed that the amino acid sequence encoded by MhPPOX1 gene was closely related to the amino acid in Pyrus bretschneideri. (2) Successfully cloning of the MhPPOX1 promoter fragment of M. halliana (2 016 bp). The New PLACE database was used to predict and analyze the cis acting elements of the promoter. There were response elements such as drought, low temperature, light, auxin and chlorophyll in MhPPOX1 promoter sequence. (3) The overexpression vector MhPPOX1pRI101 was successfully constructed, and transgenic N. tabacum L. and A. thaliana were successfully obtained. (4) qRTPCR analysis showed that under iron deficiency stress (Fe) the leaves of M. halliana seedlings turned yellow and died, and the expression level of MhPPOX1 gene was significantly higher than that of the control. Compared with wild type, under iron deficiency stress transgenic N. tabacum L. and A. thaliana grew well and not easy yellowing. The total chlorophyll a, chlorophyll b and total iron contents of transgenic N. tabacum L. and A. thaliana were significantly higher than those of wild type plants, which indicated that overexpression of MhPPOX1 gene improved the resistance of N. tabacum L. and A. thaliana to iron deficiency stress. The study suggested that MhPPOX1 gene plays an important role in plant resistance to iron deficiency stress.