The fitness effects of mutations are context specific and depend on both external (e.g., environment) and internal (e.g., cellular stress, genetic background) factors. The influence of population size and density on fitness effects are unknown, despite the central role population size plays in the supply and fixation of mutations. We addressed this issue by comparing the fitness of 92 Keio strains (Escherichia coli K12 single gene knockouts) at comparatively high (1.2×10(7) CFUs/mL) and low (2.5×10(2) CFUs/mL) densities, which also differed in population size (high: 1.2×10(8); low: 1.25×10(3)). Twenty-eight gene deletions (30%) exhibited a fitness difference, ranging from 5 to 174% (median: 35%), between the high and low densities. Our analyses suggest this variation among gene deletions in fitness responses reflected in part both gene orientation and function, of the gene properties we examined (genomic position, length, orientation, and function). Although we could not determine the relative effects of population density and size, our results suggest fitness effects of mutations vary with these two factors, and this variation is gene-specific. Besides being a mechanism for density-dependent selection (r-K selection), the dependence of fitness effects on population density and size has implications for any population that varies in size over time, including populations undergoing evolutionary rescue, species invasions into novel habitats, and cancer progression and metastasis. Further, combined with recent advances in understanding the roles of other context-specific factors in the fitness effects of mutations, our results will help address theoretical and applied biological questions more realistically.