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The lack of an efficient transformation system for Pinellia ternata is a major bottleneck in the functional annotation of genes and development of increased stress resistance of this medicinal herb. Here we describe the efficient transformation of P. ternata using petioles from submerged cultures as explants. We used the Agrobacterium strain EHA105, which harbored a pCAMBIA2300-35S-GUS-CaMVterm plasmid containing β-glucuronidase (GUS) as the reporter gene and nptII as the selectable marker. The recovery of transgenic plants was achieved by callus induction on the selection medium followed by shoot induction on the regeneration medium under selection pressure. Pre-culture of explants and ultrasonic treatment during inoculation enhanced the transformation efficiency. Transformation frequency reached 19% based on a GUS assay of independently derived, putative transgenic lines. This transformation system should facilitate the functional characterization of genes of interest and genetic advancements in P. ternata.