Carbon source, an essential nutrient for plant growth, mainly includes exogenous sugar and CO2 of the environment in vitro. Therefore, the exogenous sugar and CO2 of the environment make the important roles in tissue culture. The aim of this study is to investigate the effects of different sugar concentrations (0, 10, 15 and 30 g·L-1) on the growth of colored Zantedeschia in vitro under certain CO2 concentration and explore the optimal sugar concentration. The plantlets in vitro of colored Zantedeschia had the largest root number, root weight, and root vigor under 0 g·L-1 (sugar-free culture) treatment. And they had the largest plant height, leaf length and leaf chlorophyll content, but p oor r oot v igor under 3 0 g·L-1 sugar. This study indicated that the optimal condition for proliferation and seedling culture of colored Zantedeschia plantlets in vitro was MS medium with 30 g·L-1 sugar, and the suitable medium for rooting culture and transplanting of colored Zantedeschia was MS medium with sugar-free culture under CO2 enrichment condition.