An efficient in vitro method for rapid vegetative propagation of Bienertia sinuspersici, one of four terrestrial species of family Chenopodiaceae capable of performing C4 photosynthesis within a single cell, was developed. Cuttings of B. sinuspersici were used to examine the effects of Murashige and Skoog (MS) media strength and auxins on adventitious root formation. Half-strength MS medium was determined to be ideal for adventitious root formation in Bienertia cuttings. Although cuttings cultured in medium containing 5.0 mg/L α-naphthalene acetic acid (NAA) promoted the highest number of adventitious roots, cuttings cultured in medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) produced the longest adventitious roots and had the highest survival rate upon transplanting to soil. Histological analysis revealed variations in the root anatomy generated by the various auxins which may affect adventitious root formation and subsequent establishment of cuttings in soil. Overall, the established procedure provides a simple and cost-effective means for the rapid propagation of the single-cell C4 species B. sinuspersici.