The local variation of grain boundary atomic structure and chemistry caused by segregation of impurities influences the macroscopic properties of polycrystalline materials. Here, the effect of co-segregation of carbon and boron on the depletion of aluminum at a Σ5 (3 1 0)[0 0 1] tilt grain boundary in a α − Fe-4 at%Al bicrystal is studied by combining atomic resolution scanning transmission electron microscopy, atom probe tomography and density functional theory calculations. The atomic grain boundary structural units mostly resemble kite-type motifs and the structure appears disrupted by atomic scale defects. Atom probe tomography reveals that carbon and boron impurities are co-segregating to the grain boundary reaching levels of >1.5 at%, whereas aluminum is locally depleted by approx. 2 at.%. First-principles calculations indicate that carbon and boron exhibit the strongest segregation tendency and their repulsive interaction with aluminum promotes its depletion from the grain boundary. It is also predicted that substitutional segregation of boron atoms may contribute to local distortions of the kite-type structural units. These results suggest that the co-segregation and interaction of interstitial impurities with substitutional solutes strongly influences grain boundary composition and with this the properties of the interface.