Athyrium wardii (Hook.) is a promising herbaceous plant species for phytostabilization of cadmium (Cd) contaminated sites with large biomass and fast growth rate. However, little information is available on its tolerance mechanisms towards Cd. To further understand the mechanisms involved in Cd migration, accumulation and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in the mining ecotypes and corresponding non-mining ecotypes of A. wardii via greenhouse pot experiment. Subcellular fractionation of Cd-containing tissues demonstrated that the majority of the element was mainly located in soluble fraction in cell walls. This indicated that both the vacuoles and cell walls might be evolved the Cd tolerance mechanisms to protect metabolically active cellular compartments from toxic Cd concentrations. Meanwhile, Cd taken up by the plant existed in different chemical forms. Results showed that the majority of Cd in plant was in undissolved Cd-phosphate complexes (extracted by 2% CH3COOH), followed by water soluble Cd-organic acid complexes, Cd(H2PO4)2, pectates and protein form (extracted by deionized water and 1 M NaCl), whereas only small of Cd in roots was in inorganic form (extracted by 80% ethanol) which suggests low capacity to be transported to aboveground tissues. It could be suggested that Cd integrated with undissolved Cd-phosphate complexes in cell wall or compartmentalization in vacuole might be responsible for the adaptation of the mining ecotypes of A. wardii to Cd stress.