The magnetite ore bodies of the Paleozoic Glubochenskoe iron deposit (315.7 Mt at ~ 30.15% Fe) are located in the northern part of the Valerianovka arc zone (“Turgai belt”) within the Transuralian Megazone, Russia. They occur in calcareous-volcaniclastic rocks, exhibit layered textures, and contain primary seafloor hematite ores. The sequence of mineral formation reflects the diagenetic to metamorphic evolution of the iron ores: (i) finely dispersed hematite-1; (ii) tabular hematite-2 crystals; (iii) pseudomorphic magnetite-1 after hematite-2; (iv) zoned magnetite-2 crystals with relict hematite-2 (or magnetite-1); (v) thin oscillatory zoned magnetite-3 crystals; and (vi) magnetite-4 porphyroblasts. A gangue assemblage of Fe-rich and Fe–Mg chlorite, illite, quartz, albite, carbonates, rutile, and apatite with rare monazite, xenotime, and zircon occurs in ore and calcareous-volcaniclastic layers. The gangue clasts (volcanic glass, Ca–Mg and Ti minerals, and altered volcanic rocks) are replaced by hematite and further by magnetite. Low siderite δ13C values from layered magnetite ores (− 8.0 to − 19.5‰ PDB) indicate the presence of primary organic matter in calcareous-volcaniclastic rocks. Siderite δ18O values (6.5 to 17.4‰ SMOW) are evidence of isotopic exchange between minerals and fluids during metamorphism. Negative δ34S values for pyrite (down to − 4.5‰) likely indicate derivation of sulfur from organic matter in clastic sedimentary rocks. LA-ICP-MS analysis of zoned magnetite demonstrates highly variable Si, Al, Mg, Na, K, Ca, Ti, Mn, Rb, Y, Zr, Sr, U, and P contents, related to inclusions of gangue minerals. Elevated homogenous V and Ga contents and low contents of Sc, Co, Ni, Ge, As, Mo, Sn, and W (average < 5 ppm) indicate their incorporation into the structure of magnetite. All element contents (except V and Ga) are significantly higher in the inner zone of magnetite (magnetite-1) compared to the outer zone of magnetite (magnetite-2). The calcareous-volcaniclastic material, which was altered during submarine weathering and leaching, is proposed to be a major source of iron to form oxides. Altogether, the paragenetic sequence, and the mineralogical and geochemical data suggest the Glubochenskoe deposit can be characterized as a volcanic-sedimentary type of banded iron formation.