SEC13 homolog, nuclear pore and COPII coat complex component (SEC13) is the core component of the cytoplasmic COPII complex, which mediates material transport from the endoplasmic reticulum to the Golgi complex. Our preliminary work found that SEC13 gene was differentially expressed in dairy cows during different stages of lactation, and involved in metabolic pathways of milk synthesis such as citric acid cycle, fatty acid, starch and sucrose metabolisms, so we considered that the SEC13 might be a candidate gene affecting milk production traits. In this study, we detected the polymorphisms of SEC13 gene and verified their genetic effects on milk yield and composition traits in a Chinese Holstein cow population. By sequencing the whole coding and partial flanking regions of SEC13, we found four single nucleotide polymorphisms (SNPs). Subsequent association analysis showed that these four SNPs were significantly associated with milk yield, fat yield, protein yield or protein percentage in the first and second lactations (p ≤.0351). We also found that two SNPs in SEC13 formed one haplotype block by Haploview4.2, and the block was significantly associated with milk yield, fat yield, fat percentage, protein yield or protein percentage (p ≤ .0373). In addition, we predicted the effect of SNP on 5'region on transcription factor binding sites (TFBSs), and found that the allele A of 22:g.54362761A>G could bind transcription factors (TFs) GATA5, GATA3, HOXD9, HOXA10, CDX1 and Hoxd13; and further dual-luciferase reporter assay verified that the allele A of this SNP inhibited the fluorescence activity. We speculate that the A allele of 22:g.54362761A>G might inhibit the transcriptional activity of SEC13 gene by binding the TFs, which may be a cause mutation affecting the formation of milk production traits in dairy cows. In summary, we proved that SEC13 has a significant genetic effect on milk production traits and the identified significant SNPs could be used as candidate genetic markers for GS SNP chips development; on the other hand, we verified the transcriptional regulation of 22:g.54362761A>G on SEC13 gene, providing research direction for further function validation tests.