Human bioavailability of the flavonoid dihydrochalcones is little understood, and no evidence exists for C-glycosyl flavonoid absorption in humans. The present study uses catechol-O-methyltransferase to generate methylated metabolites of aspalathin (a C-glycosyl dihydrochalcone from rooibos tea). One of the methylated forms, both with and without glucuronidation, was detected using LC-MS/MS in the urine of human subjects (n = 6), demonstrating that deglycosylation is not a prerequisite for C-glycosyl flavonoid absorption. Methylation is catalysed by both intestine and liver cytosolic extracts. The results show that flavonoid C-glycosides are methylated and glucuronidated in vivo in an intact form in humans.