Eleocharis acicularis, an aquatic macrophyte of the Cyperaceae family, has been shown to accumulate multiple heavy metals and has great potential for use in the phytoremediation of contaminated soil and water. To investigate the mechanism of accumulation of heavy metals in E. acicularis while excluding biotic and abiotic environmental effects and to acquire homogenous and sufficient populations of E. acicularis, we established an aseptic culture system and analyzed the applicability of this species for phytoremediation. Young ramet bases and stolon tips of E. acicularis grown in the field were sterilized, cultured, and established in an aseptic culture system, and the effective growth conditions of isolated ramets were determined. Isolated ramets grew remarkably well in a medium of pH 4.8 to 5.7 with 0.25 mg/L kinetin as a plant hormone. Furthermore, capacity for the accumulation of heavy metals was examined using E. acicularis subcultured with or without Si. Aseptically cultured E. acicularis showed a sufficient capacity for Cs and Zn accumulation and exceeded the criteria for hyperaccumulator plants in accumulating Pb, Cd, and In regardless of the addition or not of Si during its subculture. The aseptic culture of E. acicularis enhances its capacity for the accumulation of heavy metals and its applicability for phytoremediation.