Sugiharto, S., Jensen, B. B., Hedemann, M. S. and Lauridsen, C. 2014. Comparison of casein and whey in diets on performance, immune responses and metabolomic profile of weanling pigs challenged with Escherichia coli F4. Can. J. Anim. Sci. 94: 479-491. The differences in amino acid composition in casein and whey have been thought to exert different immunomodulatory and metabolic effects. The objective of this study was to compare the effects of casein and whey on performance, immune responses and plasma metabolomic profile of weanling pigs challenged with Escherichia coli F4. Two factorial experiments involving 24 weanling pigs were conducted. Diets containing casein or whey, and challenge with E. coli O149:F4 or not, were the two factors. Blood was sampled on the day before challenge and 4 and 7 d postchallenge. For measurement of mucosal immune responses and intestinal dimensions, samples were collected from the small intestine (SI) immediately after killing at 7 d postchallenge. Feeding the whey diet decreased (P<0.05) mucosal immunoglobulin (Ig) A concentration and percentage of CD3 T cells in the blood, and tended (P=0.114) to upregulate the gene expression of tumor necrosis factor (TNF)-α in SI tissue, compared with feeding the casein diet. The diarrhea occurrence (P<0.05) and CD4 :CD8 T cell ratio (P=0.058) were higher, whereas the percentage of lymphocytes to total leukocytes was lower (P<0.05) in challenged pigs compared with unchallenged pigs. The challenge reduced (P<0.05) daily gain and tended to reduce the daily feed intake (P=0.067) and the intestinal mucosa weight (P=0.073), but an effect of diet was not observed. The metabolomic analysis showed discrimination between challenged and unchallenged pigs after inoculation with E. coli, but no discrimination was observed between pigs fed casein and whey diets before and after challenge. In conclusion, the feeding of casein or whey to weanling pigs differed in immunological responses, whereas no distinctions were observed with regard to performance and plasma metabolomic profile of pigs. Challenge of the pigs with E. coli F4 increased the diarrhea and appeared to modulate the immune function of the piglets through changes in T cells populations and plasma metabolite profile.