Alkaloids, a class of low-molecular-weight nitrogenous compounds, attract a great deal of interest because of their biological activities and therapeutic potential. Yet, surprisingly little is known about their interactions with drug transporters, especially Organic Anion Transporting Polypeptide 1B1 (OATP1B1), a liver-specific uptake transporter, which is closely associated with drug-induced liver injury (DILI). This study aims to investigate the inhibitory effects of 160 alkaloids on OATP1B1, assess the hepatoprotective effects against bosentan-induced liver injury, and elucidate the structure-activity relationships of alkaloids with OATP1B1. Four alkaloids, including dihydroberberine, deacetyltaxol, dihydrocapsaicin, and tetrahydropalmatine, significantly inhibited OATP1B1 transport activity in OATP1B1-HEK293 cells (>
50%), which reduced the OATP1B1-mediated uptake of methotrexate and microcystin-LR, and consequently decreased their cell toxicity. In bosentan-induced liver injury models, 4 alkaloids reduced serum total bile acid (TBA) levels and liver concentration of bosentan to different degrees, especially deacetyltaxol, which exhibited the most potent hepatoprotective effect against bosentan. The pharmacophore model suggested that the critical pharmacophores of alkaloid inhibitors are hydrogen bond acceptors and hydrophobic groups. Our findings pave the way for predicting the potential risks of alkaloids-containing food/herb-drug interactions in humans and optimizing the alkaloid structure for alleviating OATP1B1-related DILI.