Abstract: Effects of drought stress on osmotic adjustment substance and protective enzyme activity of Pinus yunnanensis seedlings were studied by the design of the different drought interval days. The results showed that within a certain range, membrane relative conductivity, malondialdehyd, proline, soluble sugar content and superoxide dismutase activity of Pyunnanensis seedlings increased with the extension of the drought interval days. But ranges of the increases were different due to the difference of the drought interval days and the type of osmotic adjustment substance. There were significantly different among treatments (drought interval days) in soluble sugar and superoxide dismutase, malondialdehyd content had great differences and no obvious difference among treatments for proline and plasma membrane relative conductivity. Osmotic adjustment ability of soluble sugar was higher than proline. Malondialdehyd has higher correlation with the drought tolerance compared with plasma membrane relative conductivity. The response of drought stress was weak for proline and plasma membrane relative conductivity. The damage of plasma membrane, osmotic adjustment and superoxide dismutase protection were carried out simultaneously, which resulted in the drought tolerance of Pyunnanensis seedlings under drought stress.