C4‐dicarboxylates, such as fumarate, l‐malate and l‐aspartate represent substrates for anaerobic growth of Escherichia coli by fumarate respiration. Here, we determined whether C4‐dicarboxylate metabolism, as well as fumarate respiration, contribute to colonization of the mammalian intestinal tract. Metabolite profiling revealed that the murine small intestine contained high and low levels of l‐aspartate and l‐malate respectively, whereas fumarate was nearly absent. Under laboratory conditions, addition of C4‐dicarboxylate at concentrations corresponding to the levels of the C4‐dicarboxylates in the small intestine (2.6 mmol kg⁻¹ dry weight) induced the dcuBp‐lacZ reporter gene (67% of maximal) in a DcuS‐DcuR‐dependent manner. In addition to its role as a precursor for fumarate respiration, l‐aspartate was able to supply all the nitrogen required for anaerobically growing E. coli. DcuS‐DcuR‐dependent genes were transcribed in the murine intestine, and mutants with defective anaerobic C4‐dicarboxylate metabolism (dcuSR, frdA, dcuB, dcuA and aspA genes) were impaired for colonizing the murine gut. We conclude that l‐aspartate plays an important role in providing fumarate for fumarate respiration and supplying nitrogen for E. coli in the mouse intestine.