The purposes of this study were to determine content uniformity of phenolics in the St. John's wort (SJW) supplements and to demonstrate how variations in the product matrices affect their absorption and efflux. LC and LC-MS/MS methods were used to determine the phenolic contents of 12 different products purchased locally or from the Internet. Three representative extracts were further submitted to Caco-2 cell transport experiment, and transport of rutin, hyperoside, and isoquercitrin was evaluated. The results indicated that the 12 different products displayed 12 different HPLC fingerprints, but all products contained the following major compounds: rutin, hyperoside, isoquercitrin, quercitrin, quercetin, and amentoflavone. The content uniformity of these major compounds was poor across products, with the smallest difference in the amounts of amentoflavone (3.6-fold) and largest difference in that of isoquercitrin (28.8-fold). The Caco-2 experiments indicated transport of rutin in products was vectorial, with the permeabilities varied about 3.6-fold in both directions of transport. The vectorial permeabilities of hyperoside and isoquercitrin were similarly different. Use of efflux transporter inhibitor studies suggested that MRP2 was involved in isoquercitrin's efflux and the product matrix affected the extent of its efflux. In conclusion, different SJW supplements had highly variable contents of phenolics, and the variability in product matrix and phytochemical compositions affected the permeabilities of key phenolics across the Caco-2 monolayers, which may further affect their bioavailabilities. Therefore, standardization will be necessary to ensure safe and efficacious using of supplements such as SJW.