During the Mississippian period, metazoan reefs and other marine faunas gradually recovered from the Late Devonian mass extinctions and reached a peak in the late Visean (~334-332 Ma). Faunal diversity started to decline from the latest Visean (~332-330 Ma) through Serpukhovian (~330-323 Ma), with significant genera/species losses and ecosystem reconstruction. This Middle-Late Mississippian biodiversity crisis (M-LMBC) was thought to have been caused by global cooling associated with the late Paleozoic Ice Age (LPIA), but existing sedimentological and temperature proxy data suggest that the global cooling event-that marks the onset of the main glaciation phase of LPIA-happened either ~4 Myr before or ~ 1-5 Myr after the initial biodiversity decline at ~332 Ma. Here, we report oxygen isotope data of diagenetically screened, well-preserved brachiopod calcite (δ¹⁸O_calcite) from late Visean-Serpukhovian (or Middle-Late Mississippian; ~334-323 Ma) strata in South China where biodiversity data are well documented. The δ¹⁸O_calcite data reveal a ~ 2.0‰ positive shift from -4.6 ± 0.2‰ to -2.7 ± 0.5‰ with an estimated ~4.7-5.5 °C drop in sea surface temperature (SST) during ~332.5-331.5 Ma in the late Visean. This cooling event coincides with fast decline of metazoan reef abundance, followed by decrease of benthic faunal diversity. The δ¹⁸O_calcite data, in combination with calibrated sedimentological and biodiversity data, demonstrate the coupling between late Visean (~332 Ma) onset of the main glaciation phase of the LPIA and initiation of the M-LMBC.