Abstract: [Objective] The Naneng gold deposit is an important medium-sized Carlin type gold deposit in southeastern Yunnan，and studying its genesis is of great significance for searching for such gold deposits in southeastern Yunnan. [Methods] Two generations of pyrite (PyI and PyII) were found to develop in the Naneng gold deposit during detailed field survey and indoor observation，and trace elements and sulfur isotopes of gold-bearing minerals are analyzed by LA-ICP-MS to constrain the source of ore-forming materials and ore genesis.[Results] LA-ICP-MS analyses show that PyI contains a small amount of Au (mean 6.37×10-6), which is relatively enriched in elements such as Co, Ni, Se, W; The distribution characteristics of trace elements in PyII and PyI are similar, but the content of Au (mean 68.02×10-6) is relatively high, and As, Sb, Cu elements are enriched in PyII; The average Au content of arsenopyrite is 36.02×10-6, and arsenopyrite is mainly enriched in elements such as As、Ni、Sb、Se、Au, while Zn, Ag, Hg and Tl elements are low. In addition, gold-bearing minerals in the Naneng gold deposit have consistent in situ δ34S values, ranging from 13.7‰ to 16.5‰, indicating that the S of gold-bearing minerals mainly come from the surrounding rocks.[Conclusion] It is preliminarily concluded that PyI was formed in a relatively stable environment by medium to low temperature hydrothermal fluid from the same source rich in trace elements such as Au、As、Sb, and a small amount of Au precipitated simultaneously with PyI in the form of solid solution (Au+). In the PyII stage, the intense tectonic activity in the area caused ore-forming fluid to upswell, and after sulfidation reaction with surrounding rock strata, the concentration of H2S in the fluid decreased, Au-HS complex became unstable, and Au supersaturated precipitation was enriched in PyII in the form of nanoscale inclusions (Au0).