Using qRT-PCR, luminol-based chemiluminescence assays, and maize protoplast subcellular localization, we evaluated the resistance of the Zmmdr1 mutant to southern corn rust and corn leaf blight and analyzed its immune responses. The results showed that in the Zmmdr1 mutant, the nucleotide at position 1997 bp of the ZmMDR1 gene was changed from G to A, resulting in premature termination of translation. After inoculation with the pathogens causing southern corn rust and corn leaf blight, the leaves of Zmmdr1 mutants showed significantly more spores and lesions compared to wild-type plants, indicating a marked reduction in resistance to both diseases in Zmmdr1 mutants. ROS levels following treatment with Flg22 were detected, and ROS production in Zmmdr1 mutants was found to be markedly lower than that in the wild-type. After pathogen inoculation, the expression of the key immune-related genes ZmrbohD and ZmPR1 was analyzed, and the expression of the two immune marker genes was found to be significantly reduced in Zmmdr1 mutants compared to the wild-type, suggesting that ZmMDR1 plays an essential role in the immune response. Subcellular localization using a maize protoplast system revealed that ZmMDR1 is a plasma membrane-localized protein.