Abstract: Micro culture environmental factors such as temperature, relative humidity, light intensity, and CO2 concentration are important to growth and development of plantlets in vitro. The aim of this study was to establish a culture process for soybean plantlet rooting under controlled environment. Single nodal soybean (Glycine max L.) explants each with 85 mg fresh mass were planted at two gas-permeable filtered culture vessels containing 70 ml medium and cultured at air temperature of 23±1℃, relative humidity of 65±5%, photosynthetic photon flux of 70±9 μmol/(m2·s), photoperiod of 16 h/d, and without CO2 control for 21 days. The medium components were MS basic ingredient and B5 medium vitamins appending six combinations of sugar (0, 20 g/L) and hormone (0.5 and 1.0 mg/L IBA; 0.5 and 1.0 mg/L NAA). The thickset rooting and growth of soybean plantlets were shown in all treatments and the treatment with 20 g/L sugar and 1.0 mg/L IBA showed greater growth variables and net photosynthetic rate than other treatments. The culture process using photoautotrophic micropropagation could be superior for soybean plantlet rooting compared to conventional tissue culture process.