While there has been much recent focus on the ecological causes of adaptive diversification, we know less about the genetic nature of the trade-offs in resource use that create and maintain stable, diversified ecotypes. Here we show how a regulatory genetic change can contribute to sympatric diversification caused by differential resource use and maintained by negative frequency-dependent selection in Escherichia coli. During adaptation to sequential use of glucose and acetate, these bacteria differentiate into two ecotypes that differ in their growth profiles. The "slow-switcher" exhibits a long lag when switching to growth on acetate after depletion of glucose, whereas the "fast-switcher" exhibits a short switching lag. We show that the short switching time in the fast-switcher is associated with a failure to down-regulate potentially costly acetate metabolism during growth on glucose. While growing on glucose, the fast-switcher expresses malate synthase A (aceB), a critical gene for acetate metabolism that fails to be properly down-regulated because of a transposon insertion in one of its regulators. Swapping the mutant regulatory allele with the ancestral allele indicated that the transposon is in part responsible for the observed differentiation between ecological types. Our results provide a rare example of a mechanistic integration of diversifying processes at the genetic, physiological, and ecological levels.