To investigate the underlying molecular mechanism of threonine (Thr) regulation on the development of breast muscle in Pekin ducks, 240 male Pekin ducks at 1 d of age were fed a Thr deficiency diet (Thr-D), Thr sufficiency diet (Thr-S), or Thr excess diet (Thr-E) for 21 d. The results showed that Thr-D reduced body weight (BW), average weight gain (ADG), and average feed intake (ADFI), and increased the feed/gain (F/G) in Pekin ducks (P < 0.05), and Thr-E did not affect BW, ADG, ADFI, or F/G (P > 0.05), compared with Thr-S. The diameter and cross-sectional area of the breast muscle fibers in the Thr-S group were larger than those in the Thr-D group (P < 0.05). RNA sequencing revealed 1,300 differential expressed genes (DEGs) between the Thr-D and Thr-S groups, of which 625 were upregulated and 675 were downregulated by Thr-D. KEGG analysis showed that the upregulated genes were enriched in mTOR, FoxO, Wnt, fat digestion and absorption, and other signaling pathways. The downregulated genes were enriched in the MAPK signaling, glycolysis/gluconeogenesis, adipocytokine signaling, and biosynthesis of unsaturated fatty acids signaling pathways. The genes of Wnt family member 3a (Wnt3a), myogenin, myozenin 2, and insulin like growth factor 2 mRNA binding protein were upregulated, and platelet derived growth factor subunit B, PDGF receptor beta and Wnt4 were downregulated by Thr deficiency, which involving in muscle development. Our findings indicated that Thr increased breast fiber size, perhaps because Thr affected the proliferation and differentiation of satellite cells in breast muscle of ducks after hatch. Our results provide novel insights into new understanding of the molecular mechanisms underlying breast muscle development in ducks subjected to dietary Thr.