The content of peptides in Shaoxing rice wine is high; however, their potential taste properties have not yet been studied. To isolate and identify bitter-tasting peptides in a popular Chinese rice wine, Shaoxing rice wine, a series of purification steps including ultrafiltration and gel filtration chromatography (GFC), combined with a taste orientation strategy, were used. According to the evaluation results of taste dilution analysis, a bitter peptide with the highest taste efficacy was isolated, and its purity was verified by high-performance liquid chromatography (HPLC). The amino acid sequence of peptide components was analyzed by ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). The bitter peptide Leu-Pro-Thr-Leu (LPTL) was successfully identified from Shaoxing rice wine. Then, taste addition experiments highlighted the bitter impacts of LPTL on sensory descriptors in Shaoxing rice wine. The LPTL peptide can contribute to bitterness in Shaoxing rice wine.