Drug resistance remains a major challenge in cancer therapy. Emerging evidence has revealed that protein lactylation, a newly discovered post-translational modification of lysine residues in histone and non-histone proteins, contributes to drug resistance in cancers. In this review, we aimed to provide an overview of the newly identified lactylation regulators (lactylation writers, erasers, and readers) and their roles and summarize the recent advances in the mechanisms by which lactylation modulates cancer drug resistance to highlight the role of lactylation in mediating cancer cell resistance to chemotherapy, immunotherapy, and targeted cancer therapy. We also aimed to provide an overview of the recent findings and emerging concepts that leverage lactylation through pharmacological inhibition to overcome drug resistance in cancers.