Synthetic biology is contributing to the advancement of the global net-negative carbon economy, with emphasis on formate as a member of the one-carbon substrate garnering substantial attention. In this study, we employed base editing tools to facilitate adaptive evolution, achieving a formate tolerance of Yarrowia lipolytica to 1 M within two months. This effort resulted in two mutant strains, designated as M25-70 and M25-14, both exhibiting significantly enhanced formate utilization capabilities. Transcriptomic analysis revealed the up-regulation of nine endogenous genes encoding formate dehydrogenases when cultivated utilizing formate as the sole carbon source. Furthermore, we uncovered the pivotal role of the glyoxylate and threonine-based serine pathway in enhancing glycine supply to promote formate assimilation. The full potential of Yarrowia lipolytica to tolerate and utilize formate establishing the foundation for pyruvate carboxylase-based carbon sequestration pathways. Importantly, this study highlights the existence of a natural formate metabolic pathway in Yarrowia lipolytica.