The skeletal muscle fiber profile is closely related to livestock meat quality. However, the molecular mechanisms determining muscle fiber types in donkeys are not completely understood. In this study, we selected the psoas major muscle (PM; mainly composed of oxidative-type muscle fibers) and biceps femoris muscle (BF; mainly composed of glycolytic-type muscle fibers) and systematically compared their mRNA and microRNA transcriptomes via RNA-seq. We identified a total of 2881 differentially expressed genes (DEGs) and 21 known differentially expressed miRNAs (DEmiRs). Furthermore, functional enrichment analysis showed that the DEGs were mainly involved in energy metabolism and actin cytoskeleton regulation. The glycolysis/gluconeogenesis pathway (including up-regulated genes such as PKM, LDHA, PGK1 and ALDOA) was more highly enriched in BF, whereas the oxidative phosphorylation pathway and cardiac muscle contraction (including down-regulated genes such as LDHB, ATP2A2, myosin-7 (MYH7), TNNC1, TPM3 and TNNI1) was more enriched in PM. Additionally, we identified several candidate miRNA–mRNA pairs that might regulate muscle fiber types using the integrated miRNA–mRNA analysis. Combined with the results of protein–protein interaction (PPI) analysis, some interesting DEGs (including ACTN3, TNNT3, TPM2, TNNC2, PKM, TNNC1 and TNNI1) might be potential candidate target genes involved in the miRNA-mediated regulation of the myofibril composition. This study is the first to indicate that DEmiRs, especially eca-miR-193a-5p and eca-miR-370, and potential candidate target genes that are mainly involved in actin binding (e.g., ACTN3, TNNT3 and TNNC1) and the glycolysis/gluconeogenesis pathways (e.g., PKM) might coregulate the myofibril composition in donkeys. This study may provide useful information for improving meat quality traits in Dezhou donkeys.