Magnetotactic bacteria perform biomineralization of intracellular magnetite (Fe3O4) nanoparticles. Although they may be among the earliest microorganisms capable of biomineralization on Earth, identifying their activity in ancient sedimentary rocks remains challenging because of the lack of a reliable biosignature. We determined Fe isotope fractionations by the magnetotactic bacteriumMagnetospirillum magneticumAMB-1. The AMB-1 strain produced magnetite strongly depleted in heavy Fe isotopes, by 1.5 to 2.5 per mil relative to the initial growth medium. Moreover, we observed mass-independent isotope fractionations in 57Fe during magnetite biomineralization but not in even Fe isotopes (54Fe, 56Fe, and 58Fe), highlighting a magnetic isotope effect. This Fe isotope anomaly provides a potential biosignature for the identification of magnetite produced by magnetotactic bacteria in the geological record.