Southern corn rust (SCR) is a destructive maize disease caused by Puccinia polysora Underw. To investigate the mechanism of SCR resistance in maize, a highly resistant inbred line, L119A, and a highly susceptible line, Lx9801, were subjected to gene mapping and transcriptome analysis. Bulked-segregant analysis coupled with whole-genome sequencing revealed several guantitative trait loci (QTL) on chromosomes 1, 6, 8, and 10. A set of 25 genes, including two coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) genes, were identified as candidate genes for a major-effect QTL on chromosome 10. To investigate the mechanism of SCR resistance in L119A, RNA-seq of P. polysora-inoculated and non-inoculated plants of L119A and Lx9801 was performed. Unexpectedly, the number of differentially expressed genes in inoculated versus non-inoculated L119A plants was about 10 times that of Lx9801, with only 29 common genes identified in both lines, suggesting extensive gene expression changes in the highly resistant but not in the susceptible line. Based on the transcriptome analysis, one of the CC-NBS-LRR candidate genes was confirmed to be upregulated in L119A relative to Lx9801 independently of P. polysora inoculation.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that transcription factors, as well as genes involved in defense responses and metabolic processes, were dominantly enriched, with the phenylpropanoid biosynthesis pathway most specifically activated. Consistently, accumulation of phenylpropanoid-derived lignin, especially S lignin, was drastically increased in L119A after P. polysora inoculation, but remained unchanged in Lx9801, suggesting a critical role of lignin in SCR resistance. A regulatory network of defense activation and metabolic change in SCR-resistant maize upon P. polysora infection is described.