The Nanmushu Zn–Pb deposit is a large-scale and representative deposit in the Mayuan ore field on the northern margin of the Yangtze Block. This study investigates the trace element geochemistry of sphalerite from this deposit using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The results show that the main trace elements in sphalerite include various trace elements, such as Mn, Fe, Cu, Ga, Ge, Ag, Cd, Pb, Co, Hg, Tl, In, Sn, and Sb. Among them, Ag, Ge, Cd, and Cu are valuable components that may be recovered during mineral processing or smelting techniques. The histograms, LA-ICP-MS time-resolved depth profiles, and linear scan profiles indicated that most trace elements occur in sphalerite as isomorphs, while partial Pb, Fe, and Ag occur as tiny mineral inclusions. The correlation diagrams of trace elements revealed that Fe2+, Mn2+, Pb2+, and Tl3+ can substitute Zn2+ in sphalerite through isomorphism. In sphalerite, Cd2+ and Hg2+ together or Mn2+, Pb2+, and Tl3+ together can replace Zn2+, i.e., ((3Mn, 3Pb, 2Tl)6+, 3(Cd, Hg)2+) ↔ 3Zn2+. Moreover, there is a mechanism of Ge4+ with Cu+ or Ga3+ with Cu+ replacing Zn2+ in the Nanmushu deposit, i.e., Ge4+ + 2Cu+↔ 3Zn2+ or 2Ga3+ + 2Cu+↔ 4Zn2+. Furthermore, the trace element compositions indicate that the Nanmushu Zn mineralization occurred under low-temperature conditions (<200 °C) and should be classified as a Mississippi Valley-type (MVT) deposit. This study provides new insights into the occurrence and substitution mechanisms of trace elements in sphalerite and the metallogenic constraints of the Nanmushu deposit.