More nuclear power plants continue to be built in China. Due to its long half-life, radiotoxicity and potential application as an environmental tracer, 236U is one of the most important artificial radionuclides deserving more study since activity data are important for risk assessment. However, the ultra-trace activity of 236U and its dilution by natural uranium isotopes make it difficult to distinguish its sources and there are only limited global fallout 236U data for present in Chinese environmental samples. In order to understand the background levels for uranium isotopes, especially 236U, and clarify their sources, inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) was applied to analyze uranium isotopes in 48 soil samples from Hunan Province, China. The 234U, 235U, 238U and 236U concentrations were measured as 9.91–33.7, 0.312–1.43, 6.63–28.7 Bq kg−1 and (1.61–21.3) × 107 atoms g−1, while, the 236U/238U, 234U/238U and 235U/238U atom ratios were (0.470–4.91) × 10−8, (5.10–9.31) × 10−5, and (7.11–7.82) × 10−3, respectively. The uranium isotopic fractionation may be due to irrigation of the agricultural lands where the samples were collected. Considering the facts that neither previous nuclear tests nor nuclear accidents had occurred in Hunan Province and the present 236U/238U atom ratios were included in the range of global fallout values in other areas, it may be concluded that 236U in soils from Hunan Province is mainly from global fallout. To the best of the authors’ knowledge, the presence of global fallout 236U in soil samples from China has been confirmed for the first time, and these values may be useful as background data for risk assessment in the future.