Since 2004, three excavations have been carried out at a late Pleistocene human fossil site of Huanglong Cave in Yunxi County, Hubei Province of China, which unearthed seven human teeth, dozens of stone tools, mammal fossils and other evidence indicating human activities. During the third excavation in 2006, in the same layer as the human teeth, we found some patches of black materials embed- ded in the deposit. We doubted that this black deposit layer is the remains of burning or even human use of fire at the cave. To further explore the possibility of human fire use at the Huanglong Cave, we examined samples directly taken from the black deposit layer and compared them with samples taken from several places in the cave using three methods: micromorphology, element content determination and deposit temperature analysis. Our results indicate that the contents of carbon element in the black deposit reach 64.59%―73.29%. In contrast, contents of carbon element of the comparative samples from other parts in the cave are only 5.82%―9.49%. The micromorphology analysis of the black deposit samples reveals a plant structure like axial parenchyma, fibrocyte, uniseriate ray and vessel. High-temperature magnetic susceptibility measurements suggest that the stratum possibly underwent a high temperature in the nature. Based on these lab analyses, we are sure that the black layer in the Huanglong Cave is the remains of fire and combustion did occur in the cave 100000 years ago. Taking other evidence of human activities found in the Huanglong Cave into consideration, we believe that the evidence of fire from the Huanglong Cave was caused by the human activities of controlled use of fire. Since 2004, three excavations have been carried out at a late Pleistocene human fossil site of Huanglong Cave in Yunxi County, Hubei Province of China, which unearthed seven human teeth, dozens of stone tools, mammal fossils and other evidence indicating human activities. During the third excavation in 2006, in the same layer as the human teeth, we found some patches of black materials embed- ded in the deposit. We doubted that this black deposit layer is the remains of burning or even human use of fire at the cave. To further explore the possibility of human fire use at the Huanglong Cave, we examined samples directly from the black deposit layer and evaluating them with samples taken from several places in the cave using three methods: micromorphology, element content determination and deposit temperature analysis. Our results indicate that the contents of carbon elements in the black deposit reach 64.59% -73.29%. In contrast, contents of carbon element of the comparative samples The micromorphology analysis of the black deposit samples reveals a plant structure like axial parenchyma, fibrocyte, uniseriate ray and vessel. High-temperature magnetic susceptibility measurements suggest that the stratum may underwent a high temperature in the nature. Based on these lab analyzes, we are sure that the black layer in the Huanglong Cave is the remains of fire and combustion did occur in the cave 100000 years ago. Taking other evidence of human activities found in the Huanglong Cave into consideration, we believe that the evidence of fire from the Huanglong Cave was caused by the human activities of controlled use of fire.