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Background: Our previous genome-wide association study (GWAS) on milk fatty acid traits in Chinese Holstein cows revealed, the SNP, BTB-01556197, was significantly associated with C10:0 at genome-wide level (P = 0.0239). It was located in the down-stream of 5-hydroxytryptamine receptor 1B (HTR1B) gene that has been shown to play an important role in the regulation of fatty acid oxidation. Hence, we considered it as a promising candidate gene for milk fatty acids in dairy cattle. In this study, we aimed to investigate whether the HTR1B gene had significant genetic effects on milk fatty acid traits., Results: We re-sequenced the entire coding region and 3000 bp of 5' and 3' flanking regions of HTR1B gene. A total of 13 SNPs was identified, containing one in 5' flanking region, two in 5' untranslated region (UTR), two in exon 1, five in 3' UTR, and three in 3' flanking region. By performing genotype-phenotype association analysis with SAS9.2 software, we observed that 13 SNPs were significantly associated with medium-chain saturated fatty acids such as C6:0, C8:0 and C10:0 (P < 0.0001 ~ 0.042). With Haploview 4.1 software, linkage disequilibrium (LD) analysis was performed. Two haplotype blocks formed by two and ten SNPs were observed. Haplotype-based association analysis indicated that both haplotype blocks were strongly associated with C6:0, C8:0 and C10:0 as well (P < 0.0001 ~ 0.0071). With regards to the missense mutation in exon 1 (g.17303383G > T) that reduced amino acid change from alanine to serine, we predicted that it altered the secondary structure of HTR1B protein with SOPMA. In addition, we predicted that three SNPs in promoter region, g.17307103A > T, g.17305206 T > G and g.17303761C > T, altered the binding sites of transcription factors (TFs) HMX2, PAX2, FOXP1ES, MIZ1, CUX2, DREAM, and PPAR-RXR by Genomatix. Of them, luciferase assay experiment further confirmed that the allele T of g.17307103A > T significantly increased the transcriptional activity of HTR1B gene than allele A (P = 0.0007)., Conclusions: In conclusion, our findings provided first evidence that the HTR1B gene had significant genetic effects on milk fatty acids in dairy cattle., (© 2021. The Author(s).)