Our previous genome-wide association study identified 83 genome-wide significant SNPs and 20 novel promising candidate genes for milk fatty acids in Chinese Holstein. Among them, the enoyl-CoA hydratase, short chain 1 (ECHS1) and enoyl-CoA hydratase and 3hydroxyacyl CoA dehydrogenase (EHHADH) genes were located near two SNPs and one SNP respectively, and they play important roles in fatty acid metabolism pathways. We herein validated whether the two genes have genetic effects on milk fatty acid traits in dairy cattle. By re-sequencing the full-length coding region, partially adjacent introns and 3000 bp up/ downstream flanking sequences, we identified 12 SNPs in ECHS1: two in exons, four in the 3 0 flanking region and six in introns. The g.25858322C>T SNP results in an amino acid replacement from leucine to phenylalanine and changes the secondary structure of the ECHS1 protein, and single-locus association analysis showed that it was significantly associated with three milk fatty acids (P = 0.0002-0.0013). The remaining 11 SNPs were found to be significantly associated with at least one milk fatty acid (P = <0.0001-0.0040). Also, we found that two haplotype blocks, consisting of nine and two SNPs respectively, were significantly associated with eight milk fatty acids (P = <0.0001-0.0125). However, none of polymorphisms was observed in the EHHADH gene. In conclusion, our findings are the first to indicate that the ECHS1 gene has a significant genetic impact on long-chain unsaturated and medium-chain saturated fatty acid traits in dairy cattle, although the biological mechanism is still undetermined and requires further in-depth validation.