Five-dimensional non-abelian gauge theories have a U(1) global symmetry associated with instantonic particles. I will describe a mixed 't Hooft anomaly between this and other global symmetries of the theory, namely the one-form center symmetry or ordinary flavor symmetry for theories with fundamental matter. I will then apply these results to supersymmetric gauge theories, analysing the symmetry enhancement patterns occurring at their conjectured RG fixed points.