Bamboo shoots, most of which are bitter in taste, are a traditional vegetable eaten in Asia. However, our understanding of the components responsible for this bitterness is limited. In this study, we used metabolomic and phytochemical analyses to scientifically study bamboo shoot bitterness. Based on oral evaluation, 16 common bamboo shoots were categorized into four bitterness groups. The most bitter species was Pleioblastus amarus. Thus, we isolated and performed a detailed spectroscopic analysis of 14 main compounds from this species. The correlation between the concentration of these components and bitterness in four representative bamboo shoot species was then analyzed by UPLC-MRM-MS and PLS-DA. It appears that bamboo shoot bitterness is largely due to L-phenylalanine, uridine, L-omithine, L-tryptophan, and adenine, with L-phenylalanine being the greatest contributor. In addition to identifying the primary component involved in bamboo shoot bitterness, this study also outlines a novel method for evaluating the bitterness of natural foods.