Finding out the genetic mechanism of growth and development traits and the development of related molecular markers can help improve the breeding of livestock. The long-chain acyl coenzyme A synthase 1 (ACSL1) gene plays a major role in lipid synthesis and fatty acid catabolism. However, there are few studies on the ACSL1 gene polymorphism of Dezhou donkeys. This study analyzed the expression level of the ACSL1 gene in different tissues of young and adult Dezhou donkeys, as well as association analysis of four gene polymorphic loci in 450 individuals. The results showed that expression levels of the ACSL1 gene are higher in heart, liver, spleen, lung, renal, gastric and muscle tissues of adult donkeys than in those of young donkeys. In the association analysis between genotype and body size traits, the wild genotype DD at the ACSL1-1 locus in female and male donkeys was greater than the mutant genotype II ( P < 0.05 ); genotype II of ACSL1-2 was significantly higher than that of DD in withers height, body length, rump width and body weight of male donkeys ( P < 0.05 ); and ACSL1-3 showed a tendency for the wild genotype II to be greater than the mutant genotype DD in female and male donkeys ( P < 0.05 ). In addition, among the five haplotype combinations constructed, Hap3Hap3 (II-II-DD-DD) and Hap6Hap6 (DD-II-II-II) haplotype combinations were superior to other haplotype combinations in growth traits, which also indicated that the results of haplotype combination association analysis and genotype association analysis tended to be the same. In conclusion, the results of this study indicate that the polymorphic loci of the ACSL1 gene can be used as candidate molecular markers for the growth and development of Dezhou donkeys, and provide a theoretical reference for the breeding of Dezhou donkeys.