Lysine lactylation, a new post-translational modification identified on histones of human and mouse cells, stimulates gene transcription from chromatin directly. However, very little is known in the scope and cellular distribution of lactylated proteins. In the present study, we conducted the first proteome-wide survey of lactylated sites in Trypanosoma brucei, a unicellular parasite causing human African sleeping sickness and livestock nagana disease, using LC-MS/MS to identify peptides enriched by immuno-purification with an anti-lactyllysine antibody. Overall, we identified 387 unique lysine lactylated sites in 257 lactylated proteins with diverse cellular localizations and biological functions. Lactylated proteins are involved in a wide variety of cellular functions such as metabolism and gene regulation. Further, we demonstrate that the lactate-derived lactylation in trypanosome is regulated by glucose metabolism. Collectively, our findings provide the first comprehensive view of the lactylome of T. brucei and suggest that lysine lactylation in trypanosomes involves a diverse array of cellular functions.