Abstract: RubisCOs were isolated and purified from Paphiopedilum micranthum, P. purpurattum, P. hirsutissimum and P. henryanum by (NH4)2SO4 precipitation, anion exchange and gel filtration chromatography in succession. The molecular weights of their large and small subunits were 51.5~54.5kD and 15~16 kD, respectively. Their zymologic characters were further studied. The results showed that these four RubisCOs from different Paphiopedilum had the highest activity at pH 8.0 and 30℃, and the descending order of RuBPcase/oase activity was P. micranthum, P. purpurattum, P. henryanum and P. hirsutissimum, and the RuBPcase/oase activity ratio of P. purpurattum and P. henryanum were higher than that of P. micranthum and P. hirsutissimum. The Km (RuBP) value of P. Micranthums RubisCO is the lowest in these four Paphiopedilum species, which indicated that its affinity to RuBP is the highest. The physiological and habitat characteristics of the four different Paphiopedilum were reflected by properties of RubisCOs isolated from them, which provided a basis for further investigation of their photosynthetic physiology.