Spontaneous Clostridium difficile (CD) outbreaks occur in neonatal piglets but the predisposing factors are largely not known. To study the conditions for CD colonisation and infection development, neonatal piglets (n=48) were moved into isolators, fed bovine milk-based formula and infected with CD 078. Analyses included: clinical scoring; faecal CD, toxin B, calprotectin; post-mortem colon histopathology. Controls were non-infected suckling piglets. Faeces from suckling, formula-fed and formula-fed-CD-infected piglets were used for metagenomics. High levels of background CD and toxin were detected in formula-fed piglets prior to infection, while suckling piglets carried about three-fold less CD, and toxin was not detected. Toxin in CD-challenged animals correlated positively with CD and calprotectin. Post-mortem signs of CDI were absent in suckling piglets, whereas mesocolonic oedema and gas-filled distal small intestines and caeca, cellular damages and reduced expression of claudins were associated with animals from the challenge trials. Microbiota in formula-fed piglets was enriched with Escherichia, Shigella, Streptococcus, Enterococcus and Ruminococcus. Formula feeding predisposes to CD colonisation earlier as compared to suckling piglets. The infection with a hypervirulent CD does not aggravate the symptoms. The association between mother and porcine milk during early life may be crucial for the control of CD expansion in piglets.