Coffee is one of the most consumed hot beverages worldwide and is highly regarded because of its stimulating effect despite having a pronounced bitterness. Even though numerous bitter ingredients have been identified, the detailed molecular basis for coffee's bitterness is not well understood except for caffeine, which activates five human bitter taste receptors. We elucidated the contribution of other bitter coffee constituents in addition to caffeine with functional calcium imaging experiments using mammalian cells expressing the cDNAs of human bitter taste receptors, sensory experiments, and in silico modeling approaches. We identified two human bitter taste receptors, TAS2R43 and TAS2R46, that responded to the bitter substance mozambioside with much higher sensitivity than to caffeine. Further, the structurally related bitter substances bengalensol, cafestol, and kahweol also activated the same pair of bitter taste receptors much more potently than the prototypical coffee bitter substance caffeine. However, for kahweol, a potent but weak activator of TAS2R43 and TAS2R46, we observed an inhibitory effect when simultaneously applied together with mozambioside to TAS2R43 expressing cells. Molecular modeling experiments showed overlapping binding sites in the receptor's ligand binding cavity that suggest that the partial agonist kahweol might be useful to reduce the overall bitterness of coffee-containing beverages. Taken together, we found that the bitterness of coffee is determined by a complex interaction of multiple bitter compounds with several human bitter taste receptors.