Alternative splicing (AS) enhances transcriptome diversity and plays important roles in regulating plant processes. Although widespread natural variation in AS has been observed in plants, how AS is regulated and contribute to phenotypic variation is poorly understood. Here, we report a population-level transcriptome assembly and genome-wide association study to identify splicing quantitative trait loci (sQTLs) in developing maize (Zea mays) kernels from 368 inbred lines. We detected 19,554 unique sQTLs for 6570 genes. Most sQTLs showed small isoform usage changes without involving major isoform switching between genotypes. The sQTL-affected isoforms tend to display distinct protein functions. We demonstrate that nonsense-mediated mRNA decay, microRNA-mediated regulation, and small interfering peptide-mediated peptide interference are frequently involved in sQTL regulation. The natural variation in AS and overall mRNA level appears to be independently regulated with different cis-sequences preferentially used. We identified 214 putative trans-acting splicing regulators, among which ZmGRP1, encoding an hnRNP-like glycine-rich RNA binding protein, regulates the largest trans-cluster. Knockout of ZmGRP1 by CRISPR/Cas9 altered splicing of numerous downstream genes. We found that 739 sQTLs colocalized with previous marker-trait associations, most of which occurred without changes in overall mRNA level. Our findings uncover the importance of AS in diversifying gene function and regulating phenotypic variation.