Changes in (total polyphenols and anthocyanins) contents, antioxidant capacity and anticancer effect, anthocyanins composition of blueberry extracts before and after in vitro digestion were investigated, and the degradation pathway of anthocyanins was speculated. Folin-Ciocalteu and pH differential methods were employed for determining the contents of total polyphenols and anthocyanins, respectively. The antioxidant capacity of the extracts before and after in vitro digestion was evaluated by the inhibition rate of liposomes, scavenging abilities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ABTS+ radical. Anthocyanin composition was analyzed using HPLC-EIS-MS/MS based on molecular mass and fragment ions, peak sequence and related literature data. The results indicated that after in vitro digestion the content of total polyphenols increased by 47.21% while anthocyanins decreased by 71.82%, and the inhibition rate of liposomes, scavenging rate of DPPH and ABTS+ free radical increased by 38.45%, 29.41% and 29.12%, respectively, and the growth inhibition of HepG2 hepatoma cells, A549 lung cancer cells and Hela human cervical cancer cells increased significantly, and the number of anthocyanins decreased from 12 to 9. Taken together, after in vitro digestion, blueberry extracts had increased total polyphenols, antioxidant activity and inhibition of the growth of the above three kinds of cancer cells, reduced anthocyanin contents and compositions. Speculation on the degradation process of gastrointestinal anthocyanins was found that the process of degradation of cyanidin-3-glucoside to form quercetin was similar to that of pelargonidin-3-glucoside degrading kaempferol.