Colloform pyrite (CPy) is widely distributed in the Tongling mineralization cluster of the Middle–Lower Yangtze River Mineralization Belt (MLYRMB), China. There have many debates as to whether such CPy is associated with Late Mesozoic igneous or Carboniferous sedimentation. CPy from the Xinqiao deposit, a representative of the stratabound sulfide deposits in the MLYRMB, was studied by powder X-ray diffraction (XRD), field-emission scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (TEM). The results show that CPy mainly comprises pyrite, pyrrhotite, quartz, and illite. Pyrite in CPy shows cubic, globule, and xenomorphic morphologies. No octahedral or pyritohedron was observed. Most of the quartz crystals display xenomorphic morphology, where pyrite mold are popular on the surface. Organic matter (OM), which is usually bound to illite, is an important component in CPy. Morphological investigations which exhibit detrital features of quartz and clay minerals indicate that they were derived from continental weathering. Specially, some hexagonal pyrrhotite nanoparticles which show mackinawite morphology are coexisted with OM. The results indicate that the transformation process of sulfides possibly is mackinawite (the precursor)—hexagonal pyrrhotite—pyrite. Thus, compositional and micro-textural characteristics of CPy in Xinqiao deposit suggest it to be a sedimentary origin rather than a hydrothermal origin which is associated with Yanshanian magmatism. Moreover, the coexistence of CPy and stratabound sulfide orebodies in the MLYRMB suggests a causal link between the two. It is considered that CPy might have served as a Cu mineralization geochemical barrier for the Cu-bearing ore-forming fluids, which originated from the Mesozoic magma in the MLYRMB.