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Rationale: Patients with type 2 diabetes may co-ingest herbal and prescription medicines to control their blood glucose. Potential interactions at uptake of compounds through P-gp, a 170 kDa ATP-dependent glycoprotein and drug transporter can affect their absorption. Furthermore, competitive binding at the catalytic sites of metabolic enzymes of drug and herb may mutually affect their metabolism. This can alter the level of drug in cell and its kinetics in the body which can lead to toxicity or loss of efficacy. Sulfonylureas are insulin secretagogues and prescribed to treat type 2 diabetes. Berberine is an active component of medicinal herbs and used by individuals to self-regulate the blood glucose levels. Around 8.5 million patients of type 2 diabetes were prescribed with sulfonylureas and 40-60% of general population use herbal medicine. A clear understanding of how absorption and metabolism of sulfonylureas (glyburide, gliclazide and glipizide) can be affected by the presence of berberine, a major chemical component of herbs used as anti-diabetics, and vice versa can provide valuable information on the potential herb drug interactions (HDIs). Methods: Berberine and sulfonylureas were co-incubated with rat liver microsomes in the presence of freshly prepared NADPH regenerating system. The metabolites of berberine and sulfonylureas were analysed using liquid chromatography with high resolution mass spectrometry operated in the positive ion mode. The role of individual isozymes involved in the metabolism of selected compounds was probed using specific inhibitors. The role of individual isozymes in metabolism of glyburide was investigated using ketoconazole and tienilic acid as the inhibitors of CYP3A and CYP2D at 0.01 to 1.5 μM and 0.005 to 100 μM, respectively, with glyburide at 20 μM in 0.5 mg/mL microsomes and the NADPH regenerating system. The role of CYP2C in metabolism of gliclazide or glipizide was investigated by incubating these substrates at 20 μM with tienilic acid (0.005 to 100 μM) in 0.5 mg/mL microsomes and the NADPH regenerating system. Cimetidine, quinidine and ketoconazole as the inhibitors of CYP1A, CYP2D and CYP3A were used at 5 to 30 μM, 0.01 to 3 μM and 0.01 to 1.5 μM, respectively with a fixed concentration of berberine of 20 μM in presence of 0.5 microsomes and the NADPH regenerating system. Potential interactions at absorption and metabolism were investigated using Caco-2 cells and microsomes, respectively. Caco-2 cells were incubated with berberine at 0.01 to 80 μM for 6 h (with or without glyburide and glipizide at 20 μM. The level of compound entering cell was measured using LC-MS. Gender differences in metabolism of berberine was also investigated by comparing the metabolic profile of the compound using human and rat liver microsomes. Results: In vitro Phase I metabolism profile of berberine was established and this confirmed the formation of demethyleneberberine (B1a) and berberrubine (B2a) with their isomers B1b and B2b, respectively. Using specific inhibitors of CYP3A and CYP2D it was found that these isozymes were found to be involved in the metabolism of berberine. In vitro Phase I metabolism of glyburide and gliclazide established the formation of hydroxylated metabolites. Inhibition studies revealed that CYP3A and CYP2C were involved in the metabolism of glyburide. Gliclazide and glipizide were metabolised by CYP2C. HDI studies revealed that glyburide affected the absorption of berberine due to the higher affinity of glyburide at the binding site of P-gp in drug binding compared with berberine in Caco-2 cells. Alternatively, berberine did not affect the absorption of glyburide. No potential interactions of glipizide and berberine was noticed to affect the absorption of either drug. A sign of potential HDIs was noticed during in vitro co-incubation of berberine with glyburide or gliclazide as the metabolism of both drugs were compromised. Glipizide did not affect the metabolism of berberine. Gender differences in the formation of berberine metabolites was observed as rat and human microsomes produced a similar profile in the in vitro biotransformation of berberine with the same metabolites formed but in different quantities. Human female microsomes were found to produce higher quantity of berberine metabolites compared to male microsomes. The rat male and female microsomes showed an inverse trend on the production of berberine metabolites. Conclusions: In vitro incubation of berberine with glyburide or gliclazide affected the metabolism of each other. These results indicate a potential risk of toxicities in the clinical practice when these compounds are used concomitantly. Potential HDIs at absorption level showed that berberine may affect the absorption of glyburide or glipizide. The interspecies and gender difference in the metabolism of berberine should be used in conjunction with other dose establishing factors to optimise the pharmacokinetics and pharmacodynamics of the drug.