At sufficiently high temperature and energy density, nuclear matter undergoes a transition to a phase in which quarks and gluons are not confined: the quark–gluon plasma (QGP)1. Such an exotic state of strongly interacting quantum chromodynamics matter is produced in the laboratory in heavy nuclei high-energy collisions, where an enhanced production of strange hadrons is observed2, 3, 4, 5, 6. Strangeness enhancement, originally proposed as a signature of QGP formation in nuclear collisions7, is more pronounced for multi-strange baryons. Several effects typical of heavy-ion phenomenology have been observed in high-multiplicity proton–proton (pp) collisions8, 9, but the enhanced production of multi-strange particles has not been reported so far. Here we present the first observation of strangeness enhancement in high-multiplicity proton–proton collisions. We find that the integrated yields of strange and multi-strange particles, relative to pions, increases significantly with the event charged-particle multipl