The recently identified histone modification lysine lactylation can be stimulated by L-lactate and glycolysis. Although the chemical group added upon lysine lactylation was originally proposed to be the L-enantiomer of lactate (K_L-la), two isomeric modifications, lysine D-lactylation (K_D-la) and N-ε-(carboxyethyl) lysine (K_ce), also exist in cells, with their precursors being metabolites of glycolysis. The dynamic regulation and differences among these three modifications in response to hypoxia remain poorly understood. In this study, we demonstrate that intracellular K_L-la, but not K_D-la or K_ce, is up-regulated in response to hypoxia. Depletion of glyoxalase enzymes, GLO1 and GLO2, had minimal impact on K_D-la, K_ce, or hypoxia-induced K_L-la. Conversely, blocking glycolytic flux to L-lactate under hypoxic conditions by knocking out lactate dehydrogenase A/B completely abolished the induction of K_L-la but increased K_D-la and Kce. We further observed a correlation between the level of K_L-la and hypoxia-inducible factor 1 alpha (HIF-1α) expression under hypoxic conditions and when small molecules were used to stabilize HIF-1α in the normoxia condition. Our result demonstrated that there is a strong correlation between HIF-1α and K_L-la in lung cancer tissues and that patient samples with higher grade tend to have higher K_L-la levels. Using a proteomics approach, we quantified 66 K_L-la sites that were up-regulated by hypoxia and demonstrated that p300/CBP contributes to hypoxia-induced K_L-la. Collectively, our study demonstrates that K_L-la, rather than K_D-la or K_ce, is the prevailing lysine lactylation in response to hypoxia. Our results therefore demonstrate a link between K_L-la and the hypoxia-induced adaptation of tumor cells.