Vitamins play an important role in maintaining normal physiologic and metabolic functions. Vitamins are known to generate bitterness, which may contribute to the off-taste of nutritional supplements. This negative sensation may decrease product acceptability and patient compliance. Few studies have examined taste thresholds and sensory sensing mechanisms at the origin of vitamin taste detection. To better understand vitamin bitterness perception, we combined human sensory analysis and taste receptor functional assays. We measured the aqueous taste detection threshold of four B vitamins using sensory difference tests. Challenging the 25 human bitter taste 2 receptors (TAS2Rs), we found that three vitamins stimulated one or more receptors. For each positive molecule-TAS2R combination, we measured the half-maximum effective concentrations (EC50). Thus, we found that the combination of sensory and biological data can provide useful explanations for the bitter detection of vitamins.