DNA-DNA interactions are important for the assembly of DNA nanostructures and during biological processes such as genome compaction and transcription regulation. In studies of these complex processes, DNA is commonly modeled as a homogeneously charged cylinder and its electrostatic interactions are calculated within the framework of the Poisson-Boltzmann equation. Commonly, a charge adaptation factor is used to address limitations of this theoretical approach. Despite considerable theoretical and experimental efforts, a rigorous quantitative assessment of this parameter is lacking. Here, we comprehensively characterized DNA-DNA interactions in the presence of monovalent ions by analyzing the supercoiling behavior of single DNA molecules held under constant tension. Both a theoretical model and coarse-grained simulations of this process revealed a surprisingly small effective DNA charge of 40% of the nominal charge density. These findings were directly supported by atomic-scale molecular dynamics simulations that determined the effective force between two DNA molecules. Our new parameterization has direct impact on many physical models involving DNA-DNA interactions.