Magnetotactic bacteria are a highly studied group of diverse prokaryotes that biomineralize chains of magnetosomes, single domain, single crystal magnetic nanoparticles of magnetite or greigite, enclosed by a lipid bilayer membrane whose synthesis is under strict genetic control. In addition to characterizing the genetics and physicochemical properties of both cultured and uncultured environmental species, there have been a number of investigations using a time course approach to determine the chemical pathway of magnetite biomineralization in these organisms. In time course studies, cells of MTB are typically grown in the absence of iron so they cannot make magnetite, and then provided with iron in culture medium which initiates the biomineralization of magnetosome chains over a subsequent time period. Results from previous time course studies are not consistent with one another, differing with regard to the nature of chemical intermediates and the rate of establishment of magnetosome chains. In this work we report a time course study of Magnetospirillum magneticum strain AMB-1 over a 48h (hour) period, using transmission electron microscopy (TEM) and soft X-ray scanning transmission X-ray microscopy (STXM) at the Fe L-edge. STXM provides capability to measure X-ray absorption spectra (XAS) and map chemical species with ∼25nm spatial resolution and thus gives detailed results on the chemistry of individual particles in single cells. An evolution of the iron oxide speciation, from a more Fe(III)-rich species, possibly α-hematite (Fe2O3), to magnetite (Fe3O4), was observed in the early stages, with evidence for the presence of the Fe(III)-rich character persisting up to 24 h. The spectromicroscopy (X-ray absorption, XAS and X-ray magnetic circular dichroism, XMCD) and TEM results show that biomineralization occurs in a stepwise fashion. First, very small particles, with no measurable magnetization, are produced at different sites in the cell without significant chain formation. The Fe L3 spectra of these early stage particles typically differ from magnetite with an additional signal at 708.4 eV that is consistent with α-hematite. By 6–8h the particles are more numerous, partial chain formation is evident, and the L3 spectrum is very similar to that of magnetite. By 24 h particles-in-chains are the dominant motif and magnetism with the moment along the chain is established. By 48 h the cells are essentially the same as cells grown in Fe-rich medium.