To date the in vitro mechanistic bioactivity of anthocyanins has been exclusively explored using aglycones and glycoside conjugates, despite a lack of evidence establishing these as the biologically available forms. We conducted intestinal epithelial cell (Caco-2 cells) culture experiments, which indicated that after a 4 h incubation of anthocyanins in cell-free culture media (DMEM), 57% of the initial cyanidin-3-glucoside (C3G) and 96% of cyanidin had degraded. The level of degradation was not statistically different from that of cultured cell incubations, suggesting that degradation was spontaneous. Degradation products were identified as protocatechuic acid (PCA) and phloroglucinaldehyde (PGA), and were confirmed in two other buffer matrices (phosphate and Hank's buffers). In cultured cell media the degradation products PCA and PGA were metabolised to glucuronide and sulphate conjugates, as indicated by both enzyme hydrolysis (sulphatase and glucuronidase treatments) and MS (PCA and PGA m/z = 155; sulphate = 235; glucuronide = 331). These data suggest a significant proportion of intestinal metabolites of anthocyanins are likely to be conjugates of their degradation products. Future efforts to establish the biological activities of anthocyanins should therefore include the investigation of phenolic acid and aldehyde products of degradation, along with their respective metabolites.