A series of novel berberine derivatives, 4,7,12,12a-tetrahydro-5H-thieno[3',2':3,4]pyrido[1,2-b]isoquinolines was designed, synthesized, and biologically evaluated for their anti-diabetic activity. Following the evaluation in two types of cells, compounds 4aa, 4bq, and 4bv stimulated glucose consumption (1.8- to 2.3-fold), reduced gluconeogenesis (60-85%), inhibited mitochondria respiratory chain complex I and activated AMPK indirectly. In a db/db mice model, compounds 4bq and 4bv lowered fasting blood glucose at a dose of 120 mg/kg/day. In addition, compounds 4bq and 4bv were found to possess improved pharmacokinetic profiles (bioavailability 45 and 106%, respectively) compared to berberine. Compounds 4bq and 4bv exhibited no obvious hERG inhibition (IC50 > 10 μM).