An in vivo/in vitro ileal fermentation assay using growing pigs has been developed but not yet formally validated. This study aimed to validate the in vivo/in vitro ileal fermentation assay by comparing in vitro fermentation values with those obtained in vivo in growing pigs. The effect of raising pigs under different environmental conditions was also investigated. Thirty piglets (1.59 ± 0.31 kg bodyweight, mean ± SD) were subjected to one of three treatments: artificially reared (non-farm, laboratory housing conditions) from postnatal day (PND) 7 (AR group), inoculated orally with human infant fecal extracts from birth until PND 8 and artificially reared (AR+ group), or conventionally reared on a farm (Control group). Starting at PND 7, the AR and AR+ pigs received human infant formula for three weeks, followed by a human-type diet for five weeks. Control pigs were weaned on the farm and, on PND 63, relocated to the laboratory animal facility. From PND 63, all pigs received a human-type diet. On PND 78, pigs were euthanized, after which ileal digesta were collected to perform an in vitro ileal fermentation (in vitro organic matter (OM) fermentability and organic acid production) and to determine digesta microbial composition, and dietary OM fermentability in vivo. The rearing regimen resulted only in a few differences in ileal microbial taxonomic composition. The rearing regimen generally did not affect the in vitro production of individual organic acids. The in vivo and in vitro OM fermentability of proximal ileal digesta (19.7 ± 2.04% on average) was similar (P > 0.05) for the AR and Control pigs but not for the AR+ pigs. The Control-rearing regimen was preferred over AR or AR+ due to ease of implementation. The in vitro ileal fermentation assay accurately predicted the in vivo OM fermentability.