The Guadalupian mass extinction took place during the major global environmental changes during Phanerozoic time. Large-scale sea-level fluctuations and a negative shift of δ13C were associated with this crisis. However, the diagenetic or primary origin of the decreased δ13C across the Guadalupian–Lopingian (G–L) boundary and the potential causes for this biotic crisis are still being intensely debated. Integrated analyses, including detailed petrographic examination, identification of foraminifer and fusulinid genera, and analysis of carbonate δ13Ccarb and bulk δ13Corg across the G–L boundary were therefore carried out at Tianfengping, Hubei Province, South China. Our results show that: (1) some foraminifer and most fusulinid genera disappear in the upper Maokou Formation (upper Guadalupian); (2) the negative shift of δ13Ccarb in the uppermost Maokou Formation is of diagenetic origin, but the values of δ13Ccarb in the remainder of the Maokou Formation and in the Wuchiaping Formation represent a primary signal of coeval seawater; and (3) the bulk δ13Corg perturbation across the G–L boundary at Tianfengping is mainly controlled by organic matter (OM) source, that is, terrestrial OM contribution. We suggest that the δ13Ccarb negative shift in the lower Wuchiaping Formation (Wuchiapingian) compared to that in the lower–middle Maokou Formation (Capitanian) were probably caused by the re-oxidization of 12C-rich OM during regression. Global regression resulted in the negative shift of δ13Ccarb at the G–L boundary in South China and led to the loss of shallow-marine benthic habitat. Large-scale global regression is probably one of the main causes for this bio-crisis.