Formate has great potential as a sustainable feedstock for biological production of biofuels. Nevertheless, utilization of formate by microbial species is limited due to its toxicity or the lack of a metabolic pathway. Here, we constructed a formate assimilation pathway in Escherichia coli and applied adaptive laboratory evolution to improve formate utilization as a carbon source in sugar-free conditions. The genes related to the tetrahydrofolate and serine cycles from Methylo-bacterium extorquens AM1 were overexpressed for formate assimilation, which was proved by the 13C-labeling experiments. The amino acids detected by GC/MS showed significant carbon labeling due to biomass production from formate. Then, 150 serial subcultures were performed to screen for evolved strains with improved ability to utilize formate. The genomes of evolved mutants were sequenced and the mutations were associated with formate dehydrogenation, folate metabolism, and bioflm formation.