DNA polymerase θ (Pol θ) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol θ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol θ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4'-oxidation (pC4-AP). Covalent modification of Pol θ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol θ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol θ and suggests a path forward for designing inhibitors of this attractive anticancer target.