Flaxseed oil is rich in ɑ-linolenic acid (ALA), which is the metabolic precursor of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). This study investigated the effect of flaxseed oil supplementation on lipopolysaccharide (LPS)-induced muscle atrophy and carbohydrate oxidation impairment in a piglet model. Twenty-four weaned pigs were used in a 2 × 2 factorial experiment including dietary treatment (5% corn oil vs. 5% flaxseed oil) and LPS challenge (saline vs. LPS). On day 21 of treatment, the pigs were injected intraperitoneally with 100 μg/kg BW LPS, or sterile saline. At 4 h after injection, blood, gastrocnemius muscle and longissimus dorsi (LD) muscle were collected. Flaxseed oil supplementation increased ALA, EPA, total n-3 polyunsaturated fatty acids contents, protein/DNA ratio, and pyruvate dehydrogenase complex (PDC) quantity in muscles (p<0.05). In addition, flaxseed oil reduced mRNA expression of toll-like receptor (TLR) 4 and nucleotide-binding oligomerization domain protein (NOD) 2 and their downstream signaling molecules in muscles, and decreased plasma concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8, and mRNA expression of TNF-α, IL-1β and IL-6 (p<0.05). Moreover, flaxseed oil inclusion increased the ratios of phosphorylated Akt 1/ total Akt 1 and phosphorylated forkhead Box O (FOXO) 1/ total FOXO1, and reduced mRNA expression of FOXO1, muscle RING finger (MuRF) 1, and pyruvate dehydrogenase kinase (PDK) 4 in muscles (p<0.05). These results suggest that flaxseed oil might have a positive effect on alleviating muscle protein loss and carbohydrates oxidation impairment induced by LPS challenge through regulation of TLR4/NOD and Akt/FOXO signaling pathway.