The Nayongzhi Zn-Pb deposit, located in the southeastern margin of the Sichuan-Yunnan-Guizhou (S-Y-G) Zn-Pb metallogenic province, China, has been recently discovered in this region and has an estimated resource of 1.52 Mt of metal at average grades of 4.82 wt % Zn and 0.57 wt % Pb. The ore bodies are hosted in the Lower Cambrian Qingxudong Formation dolostone and occur as stratiform, stratoid and steeply dipping veins. The predominant minerals are sphalerite, galena, dolomite, calcite with minor pyrite, and barite. In this paper, the inductively coupled plasma mass spectrometry (ICP-MS) technique has been used to investigate the concentrations of Fe, Cd, Ge, Ga, Cu, Pb, Ag, In, Sn, Sb, Co and Mn in bulk grain sphalerite from the Nayongzhi deposit, in an effort to provide significant insights into the element substitution mechanisms, ore-forming temperature and genesis of the deposit. This study shows that those trace elements (i.e., Cd, In, Sn, Sb, Fe, Mn, Cu, Ga, Ge, Ag, and Co) are present in the form of isomorphism in sphalerite, and strong binary correlation among some elements suggests direct substitution as Zn2+Fe2+ and coupled substitutions as Zn2+Ga3+ + (Cu, Ag)(+) and Zn2+In3+ + Sn3+ + (vacancy), despite there being no clear evidence for the presence of Sn3+. Sphalerite from the Nayongzhi deposit is enriched in Cd, Ge and Ga and depleted in Fe, Mn, In and Co, which is similar to that of the Mississippi Valley-type (MVT) deposit and significantly different from that of the Volcanogenic Massive Sulfide (VMS) deposit, Sedimentary-exhalative (Sedex) deposit, skarn, and epithermal hydrothermal deposit. Moreover, the ore-forming temperature is relatively low, ranging from 100.5 to 164.4 degrees C, as calculated by the GGIMFis geothermometer. Geological characteristics, mineralogy and trace element contents of sphalerite suggest that the Nayongzhi deposit is a MVT deposit. In addition, according to the geological characteristics, Ag content in sphalerite, and Pb isotope evidence, the Nayongzhi deposit is distinct from the deposits associated with the Indosinian Orogeny in S-Y-G Zn-Pb metallogenic province (e.g., Huize, Daliangzi, Tianbaoshan and Tianqiao deposits), thus, suggesting that multi-stage Zn-Pb mineralization may have occurred in this region.