Commercial doubled haploid (DH) breeding has been widely adopted in maize, making it essential to elucidate the spontaneous haploid genome doubling (SHGD) capacity of parental inbred lines. This study aimed to dissect the molecular mechanisms underlying SHGD in maize by comparing a high-doubling line (RL36Hap) with a low-doubling line (HCL645Hap). Anther morphology, I?-KI staining, transcriptome sequencing, and qPCR were employed to compare gene expression profiles at two developmental stages (1.0 and 1.5). RL36Hap exhibited normal anther development and 95% pollen viability, whereas HCL645Hap showed shriveled anthers and only 0.2% pollen viability. Transcriptome analysis identified differentially expressed genes (DEGs), with 6,345 detected in HCL645Hap compared with 2,987 in RL36Hap. Gene Ontology (GO) enrichment analysis showed that HCL645Hap was significantly enriched in microtubule-based movement, microtubule motor activity, DNA replication initiation, cytoplasmic translation, and structural constituents of ribosomes, whereas RL36Hap was enriched in nucleosome assembly, β-amylase activity, response to heat, and cell wall organization. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed significant enrichment of ribosome, DNA replication, fatty acid metabolism, and riboflavin metabolism pathways in HCL645Hap, whereas phenylpropanoid biosynthesis and plant–pathogen interaction pathways were enriched in RL36Hap. Heatmap analysis and qPCR validation indicated that microtubule- and DNA replication-related genes were aberrantly overexpressed and dysregulated in HCL645Hap, whereas RL36Hap maintained coordinated expression at moderate levels. Collectively, differences in SHGD capacity are associated with a trade-off among microtubule dynamics, redox homeostasis, and defense responses, and moderate rather than excessive regulation appears critical for efficient spontaneous genome doubling.