A total of 24 weaned piglets (8.53 ± 1.00 kg) were used to investigate the effects of chitooligosaccharide (COS) supplementation on immunological responses challenged with E. coli lipopolysaccharide (LPS). Experimental treatments were arranged in a 2 × 2 factorial design (n = 6/group), with the main effects of COS (0 vs. 300 mg/kg) and LPS challenge (LPS vs. saline). Piglets were raised individually in metabolic cages and fed 0 or 300 mg/kg COS for 18 d. On d 15~17, piglets were challenged with LPS (80 μg/kg BW) or saline daily. Blood was obtained at 3 h and 48 h post-injection on d 17. At 48 h post-injection, weight of spleen was recorded, thymus and spleen were sampled. All data were analyzed by 2-way ANOVA using GLM procedures of SAS, including COS, LPS, and their interaction as the fixed effects. Spleen index was elevated by LPS challenge (+37.88%, P < 0.01), without an effect of COS supplementation. Serum TNF-α (+16.99%, P < 0.05) concentrations increased and IL-10 (−11.61%, P < 0.01) decreased in pigs injected with LPS at 3 h after injection. Serum IL-1 β, IL-2 and IL-4 content were increased (+5.58%, +12.69% and +11.77%, P < 0.05) at 48 h after injection. Piglets supplemented with COS had lower serum IL-1 β and IL-2 at 3 h (−48.36% and −12.14%, P < 0.05) post-injection, and lower serum IL-6 at 3 and 48 h post-injection (−10.43% and −13.13%, P < 0.05). However, COS supplementation increased serum IL-10 at 3h post-challenge compared with non-supplemented pigs (+8.44%, P < 0.01). These alterations in serum inflammatory factor traits in piglets challenged by LPS were accompanied by increased gene expressions of CD14, MyD88 in spleen (+12.63% and +26.77%) and TLR4, NF-kB, TRIF, IRF3 in thymus (+245.81%, +44.44%, +17.13% and +86.55%) that are related to MyD88 dependent and independent signaling pathway (P < 0.05), while COS supplementation alleviated most of these changes (−37.23% for CD14, −34.87% for MyD88, −30.43% for NF-kB, −24.30% for TRIF and −31.82% for IRF3, P < 0.05). In conclusion, results indicated that COS supplementation attenuated the immune challenge of LPS possibly by inhibiting over-activation of TLR4-MyD88 dependent and independent signaling pathway.