Glucosinolates, phytochemicals found in cruciferous vegetables, are metabolised to bioactive isothiocyanates (ITC) by certain bacteria in the human gut. Substantial individual variation in urinary ITC excretion has been observed in previous cruciferous vegetable-feeding studies. We hypothesised that individual differences in gut microbial community contribute to the observed variation in glucosinolate metabolism, i.e. gut microbiota composition between high- and low-ITC excreters differs. We recruited twenty-three healthy individuals and fed them a standardised meal containing 200 g of cooked broccoli. After the meal, 24 h urinary ITC excretion was measured. Study participants with the highest (n 5) and lowest (n 5) ITC excretion provided faecal samples for ex vivo bacterial cultivation with 50 μm-glucoraphanin, the major glucosinolate found in broccoli. When grown ex vivo, faecal bacteria from the selected high-ITC excreters were able to degrade more glucoraphanin than those from the low-ITC excreters (P = 0·05). However, bacterial fingerprints of faecal and ex vivo culture microbiota revealed no statistically significant differences between the high- and low-ITC excreters in terminal restriction fragment length polymorphism analysis of the bacterial 16S ribosomal RNA gene. In conclusion, glucosinolate degradation by faecal bacteria ex vivo may be associated with in vivo bacterial glucosinolate metabolism capacity, but no direct link to specific bacterial species could be established, possibly due to the complexity and functional redundancy of the gut microbiota.