Abstract: In order to investigate the effects of different Ca²⁺ concentrations（0, 1.5, 3, 6, 9 mmol/L） on stomatal cells, agronomic characters and enzyme activities of tissue culture seedlings of Anoectochilus roxburghii（Wall.） Lindl, tissue culture plantlets of A. chinensis were used as experimental materials. The results showed that, in 1.5 mmol/L Ca²⁺ environment, the leaves of Anoectochilus roxburghii（Wall.） Lindl were broad, the plants are relatively fat, and the overall growth was good. Physiological and biochemical indicators showed that the metabolic activities of Anoectochilus roxburghii（Wall.） Lindl at this concentration were vigorous, the activities of various key enzymes were relatively high, and the stress resistance was better. Under the condition of 6 mmol/L Ca²⁺, the leaves were small and young, but the plants had many and strong plexus buds. Therefore, 1.5 mmol/L Ca²⁺ was a relatively suitable calcium environment for the growth and development of tissue culture seedlings, while 6 mmol/L Ca²⁺ was a relatively suitable calcium environment for the growth and propagation of long buds of Anoectochilus roxburghii（Wall.） Lindl.