© 2016 The Author(s). Background: This study aimed to investigate the possible serum protein changes after endotoxin administration in healthy and choline-treated calves using proteomics. These results are expected to contribute to the understanding of the pathophysiological mechanisms of endotoxemia and the beneficial effect of choline administration in this clinical situation. Methods: Healthy-calves (n = 20) were divided into 4 groups: Control, Choline treated (C), Lipopolysaccharide administered (LPS), and LPS + C. Control calves received 0.9 % NaCl injection. Calves in C and LPS + C groups received choline chloride (1 mg/kg/iv). Endotoxin (LPS) was injected (2 μg/kg/iv) to the calves in LPS and LPS + C groups. Serum samples were collected before and after the treatments. Differentially expressed proteins (> 1.5 fold-change relative to controls) were identified by LC-MS/MS. Results: After LPS administration, 14 proteins increased, and 13 proteins decreased within 48 h as compared to controls. In the LPS group, there were significant increases in serum levels of ragulator complex protein (189-fold) and galectin-3-binding protein (10-fold), but transcription factor MafF and corticosteroid binding globulin were down regulated (≥ 5 fold). As compared with the LPS group, in LPS + C group, fibrinogen gamma-B-chain and antithrombin were up-regulated, while hemopexin and histone H4 were down-regulated. Choline treatment attenuated actin alpha cardiac muscle-1 overexpression after LPS. Conclusions: LPS administration produces changes in serum proteins associated with lipid metabolism, immune and inflammatory response, protein binding/transport, cell adhesion, venous thrombosis, cardiac contractility and blood coagulation. The administration of choline is associated with changes in proteins which can be related with its beneficial effect in this clinical situation.