We introduce Seshadri constants for line bundles in a relative setting. They generalize the classical Seshadri constants of line bundles on projective varieties and their extension to vector bundles studied by Beltrametti-Schneider-Sommese and Hacon. There are similarities to the classical theory. In particular, we give a Seshadri-type ampleness criterion, and we relate Seshadri constants to jet separation and to asymptotic base loci. We give three applications of our new version of Seshadri constants. First, a celebrated result of Mori can be restated as saying that any Fano manifold whose tangent bundle has positive Seshadri constant at a point is isomorphic to a projective space. We conjecture that the Fano condition can be removed. Among other results in this direction, we prove the conjecture for surfaces. Second, we restate a classical conjecture on the nef cone of self-products of curves in terms of semistability of higher conormal sheaves, which we use to identify new nef classes on self-products of curves. Third, we prove that our Seshadri constants can be used to control separation of jets for direct images of pluricanonical bundles, in the spirit of a relative Fujita-type conjecture of Popa and Schnell.