The oxygen isotopic values of aquatic plant cellulose and carbonates in Lake Caohai sediments were measured using a continuous flow isotopic ratio mass spectrometer (CF-IRMS). Because of predictable oxygen isotopic fractionation between cellulose and its source water, the oxygen isotopic composition of paleo-lake water has been established quantitatively. Combined oxygen isotopic values of cellulose and carbonates were used in the ‘Craig’ equation to determine paleotemperatures and their variation in the lake during the past 500 years. Results show that the paleotemperature trend correlates well with meteorological records from Weining. There are four notable cold intervals at Lake Caohai over the past 500 years, namely 1540–1570AD, 1670–1715AD, 1780–1870AD and 1900–1930AD, and the former three cold intervals have been observed in the conventional Little Ice Age (LIA). These cold periods at Lake Caohai correspond well with those recorded from tree ring, peat, and ice core data from adjacent regions, particularly temperature those inferred from δ18O of peat cellulose from Hongyuan Southwestern China. The trend in paleotemperature variations at Lake Caohai are also consistent with both the change of Indian summer monsoon, derived from δ18O values of a stalagmite in Dongge, and a recorded shift in solar activity. The findings of this study illustrate that coupled analysis of δ18O values of cellulose and carbonates from lake sediments may be used as a paleotemperature proxy. These results also provide further evidence of the existence of LIA in southwestern China.