The Wutonggou iron deposit is located in the well-known iron metallogenic belt in the eastern Tianshan, NW China, and has been regarded as a sedimentary iron deposit. Hydrothermal overprinting could play indispensable roles in the formation of high-grade iron ores in sedimentary iron deposits, thus the nature and evolution of hydrothermal fluids have important implications on the genesis of these deposits. Consequently, an integrated study of ore geology, H–O–C isotopes and 40Ar–39Ar dating, is conducted on the Wutonggou deposit, in order to reveal the features, source, and timing of hydrothermal mineralization. The studied deposit includes two mining sections namely the Jianshan and Wutonggou. The δ18O values of early magnetite from the Jianshan section range from +3.0‰ to +5.8‰ that nearly consistent with classic magmatic magnetite, while increase to 6.3‰–8.0‰ in the late stage. Quartz from the two sections shows comparable H–O isotopic compositions and identical fractionation trends, and is plotted in or periphery to the primary magmatic water area. Calcites from the two sections are broadly similar in carbon and oxygen isotopic compositions, and siderite from the Wutonggou section is plotted in the same region. Thus, comparable stable isotopic compositions and evolution trends indicate similar magmatic fluids contributed hydrothermal iron mineralization in the two mining sections. Moreover, water-rock interactions of varying degrees generated distinct mineralization styles in the Jianshan and Wutonggou sections, and caused the isotopic fractionation in late stages. Biotite extracted from a hydrothermal siderite ore yielded a 40Ar–39Ar plateau age of 299.5 ± 2.0 Ma, indicating the timing of hydrothermal iron mineralization is corresponding to the emplacement of vicinity granitoids. Taken together, the hydrothermal mineralization in the Wutonggou iron deposit was the product of remobilization and upgrading of early sedimentary iron ores, and ore-forming fluids were most probably originated from regional granitic magmatism.