The Langwashan Fe deposit, with 80 million tons of proven iron ore reserves, is located in the eastern portion of the Tianshan orogenic belt, NW China. Previous research carried out detailed geological, geochemical, and geophysical studies on the deposit, while there are still different views on the ore genesis. In this study, electron probe micro-analysis and laser ablation (multi-collector) inductively coupled plasma mass spectrometry analysis of variable minerals in ore samples were conducted. Based on ore texture, in-situ biotite RbSr dating, magnetite and pyrite composition, and mineral assemblage, the Langwashan Fe mineralization has been divided into two metallogenic periods: volcanic hydrothermal metallogenic period (similar to 344 Ma) and skarn metallogenic period (similar to 239 Ma). The Early Carboniferous mineralization is characterized by layered ore bodies, a narrow range of delta S-34 values (+1.37 to +2.77 parts per thousand), and low Co/Ni ratio in early pyrite (Py I), and high Ti, V, Cr, Ni, and Co contents in early magnetite (Mag I), showing single and deep origin for the fluid. In contrast, the Early Triassic mineralization has irregular-shaped ore bodies, higher contents of Si, Al, Mn, Mg, and Ca in late magnetite (Mag II), higher Co, Ni, As, and Cu contents, and a wider range of delta S-34 values (-0.18 to +3.35 parts per thousand) in late pyrite (Py II), indicating mixed origin for the fluid. Thus, we propose that the Langwashan Fe deposit is formed by the superposition of two types of mineralization. The Early Carboniferous Hongshishan inter-arc oceanic basin subducted beneath the southern Tarim plate, and the iron-bearing magma migrated along the volcanic channel and annular faults, forming the first period of layered Fe ore bodies together with the volcanic rock deposition. During the Early Triassic, a hidden intrusion was generated in an extensional environment in the region, interacting with surrounding rocks through metasomatism, forming superimposed skarn-type Fe ore bodies. The two periods of mineralization determined in Langwashan have great significance for further exploration and prospecting of Fe ore deposits in the Tianshan orogenic belt.