Theacrine, a purine alkaloid derived from Camellia assamica var. kucha, has a distinct bitter taste. Our previous study found the lower recognition threshold of theacrine at 25 °C than 45 °C. This study aims to investigate the bitterness characterizations of theacrine at aforementioned temperatures and its taste perception mechanism. Sensory analysis exhibited higher bitterness intensity for theacrine at 25 °C than 45 °C. Subsequently, flow cytometry was performed to verify the above characterization at the cellular level. It revealed that theacrine could activated the bitter receptor hTAS2R14 and the calcium signal at 25 °C was higher than 45 °C. Ultimately, the interaction mechanism was studied by molecular dynamics simulations, indicating that the conformation of theacrine-hTAS2R14 had a higher binding capacity and better stability at 25 °C. Overall, temperature affected the binding of theacrine to the bitter receptor hTAS2R14, resulting in the stronger bitterness intensity of theacrine at 25 °C than 45 °C.