We carry out the non-perturbative renormalization of the chromo-magnetic operator in Heavy Quark Effective Theory. At order 1/m of the expansion, the operator is responsible for the mass splitting between the pseudoscalar and vector B mesons. We obtain its two-loop anomalous dimension in a Schrodinger functional scheme by successive one-loop conversions to the lattice MS scheme and the scheme. We then compute the scale evolution of the operator non-perturbatively in the Nf = 0 theory between μ ≈ 0.3 GeV and μ ≈ 100 GeV, where contact is made with perturbation theory. The overall renormalization factor that converts the bare lattice operator to its renormalization group invariant form is given for the Wilson gauge action and two standard discretizations of the heavy-quark action. As an application, we find that this factor brings the previous quenched predictions of the B*–B mass splitting closer to the experimental value than found with a perturbative renormalization. The same renormalization factor is applicable to the spin-dependent potentials of Eichten and Feinberg.