Increasing maize kernel starch content may not only lead to higher maize kernel yields and qualities, but also help meet industry demands. By using the intermated B73 × Mo17 population, QTLs were mapped for starch content in this study. A major QTL Qsta9.1 was detected in a 1.7 Mb interval on chromosome 9 and validated by allele frequency analysis in extreme tails of a newly constructed segregating population. According to genome-wide association study (GWAS) based on genotyping of a natural population, we identified a significant SNP for starch content within the ORF region of GRMZM5G852704_T01 colocalized with QTL Qsta9.1. Co-expression network analysis was also conducted, and 28 modules were constructed during six seed developmental stages. Functional enrichment was performed for each module, and one module showed the most possibility for the association with carbohydrate-related processes. In this module, one transcripts GRMZM2G110929_T01 located in the Qsta9.1 assigned 1.7 Mb interval encoding GLABRA2 expression modulator. Its expression level in B73 was lower than that in Mo17 across all seed developmental stages, implying the possibility for the candidate gene of Qsta9.1. Our studies combined GWAS, mRNA profiling, and traditional QTL analyses to identify a major locus for controlling seed starch content in maize.