ABSTRACT: Genome-wide association studies (GWASs) have been widely used to determine the genetic architecture of quantitative traits in dairy cattle. In this study, with the aim of identifying candidate genes that affect milk protein composition traits, we conducted a GWAS for nine such traits (?s1-casein, ?s2-casein, ?-casein, ?-casein, ?-lactalbumin, ?-lactoglobulin, casein index, protein percentage, and protein yield) in 614 Chinese Holstein cows using a single-step strategy. We used the Illumina BovineSNP50 Bead chip and imputed genotypes from high-density single-nucleotide polymorphisms (SNPs) ranging from 50 to 777 K, and subsequent to genotype imputation and quality control, we screened a total of 586,304 informative high-quality SNPs. Phenotypic observations for six major milk proteins (?s1-casein, ?s2-casein, ?-casein, ?-casein, ?-lactalbumin, and ?-lactoglobulin) were evaluated as weight proportions of the total protein fraction (wt/wt%) using a commercial enzyme-linked immunosorbent assay kit. Informative windows comprising five adjacent SNPs explaining no < 0.5% of the genomic variance per window were selected for gene annotation and gene network and pathway analyses. Gene network analysis performed using the STRING Genomics 10.0 database revealed a co-expression network comprising 46 interactions among 62 of the most plausible candidate genes. A total of 178 genomic windows and 194 SNPs on 24 bovine autosomes were significantly associated with milk protein composition or protein percentage. Regions affecting milk protein composition traits were mainly observed on chromosomes BTA 1, 6, 11, 13, 14, and 18. Of these, several windows were close to or within the CSN1S1, CSN1S2, CSN2, CSN3, LAP3, DGAT1, RPL8, and HSF1 genes, which have well-known effects on milk protein composition traits of dairy cattle. Taken together with previously reported quantitative trait loci and the biological functions of the identified genes, we propose 19 novel candidate genes affecting milk protein composition traits: ARL6, SST, EHHADH, PCDHB4, PCDHB6, PCDHB7, PCDHB16, SLC36A2, GALNT14, FPGS, LARP4B, IDI1, COG4, FUK, WDR62, CLIP3, SLC25A21, IL5RA, and ACADSB. Our findings provide important insights into milk protein synthesis and indicate potential targets for improving milk quality.