Dietary threonine (Thr) deficiency enhances triglyceride (TG) deposition in the liver of Pekin ducks, which injures hepatic function and impairs growth performance. However, the underlying molecular mechanisms remain unclear. In the present study, we investigated the effects of dietary Thr deficiency on the expressions of proteins and phosphoproteins in liver of Pekin ducks, to identify the underlying molecular changes. A total of 300 one-day-old ducklings were divided into 3 groups with 10 replicates of 10 birds. All ducks were fed corn-wheat-peanut meal diets containing 0.46%, 0.71%, and 0.96% Thr, respectively, from 1 to 21 days of age. Growth performance, serum parameters, hepatic TG content, and expression of genes involved in lipid metabolism of Pekin ducks were determined. A Thr deficiency group (Thr-D, 0.46% Thr) and a Thr sufficiency group (Thr-S, 0.71% Thr) were selected for subsequent proteomic and phosphoproteomic analysis. The results showed that Thr-D reduced the growth performance (P < 0.001), and increased the plasma concentrations of cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and hepatic TG (P < 0.05). Thr-D increased gene expression related to fatty acid and TG synthesis (P < 0.05). A total of 176 proteins and 259 phosphosites (containing 198 phosphoproteins) were observed to be differentially expressed as a result of Thr-D. The upregulated proteins were enriched in the pathway related to amino acid metabolism, peroxisome. The downregulated proteins were enriched in linolenic and arachidonic acid metabolism, and the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. The upregulated phosphoproteins were enriched in the pathways related to fatty acid biosynthesis, fructose and mannose metabolism, and glycolysis/gluconeogenesis. Thr-D reduced the phosphorylation of STAT1 at S729 and STAT3 at S728, and expression of STAT5B. In contrast, Thr-D increased non-receptor tyrosine-protein kinase (TYK2) expression and STAT1 phosphorylation at S649. Taken together, dietary Thr-D increased hepatic TG accumulation by upregulating the expression of genes and proteins, and phosphoproteins related to fatty acid and triglyceride synthesis. Furthermore, these processes might be regulated by the JAK-STAT signaling pathway, especially the phosphorylation of STAT1 and STAT3.