As its tuberous alkaloids induce valuable pharmacological effects, Pinellia ternata has considerable clinical value. However, its production currently fails to meet its demand. In vitro microtuber culture, combined with salicylic acid (SA) elicitation, may provide an effective alternative to traditional field production. In this study, an in vitro P. ternata microtuber induction system was developed and used to evaluate SA-induced elicitation of alkaloid accumulation. The quality of in vitro microtubers was assessed by total alkaloid measurement, a cytotoxicity assay, and transcriptomic analysis. With or without SA treatment, P. ternata microtuber induction was achieved within 60 d using petiole-derived explants, with a microtuber proliferation rate of approximately 17 microtubers per explant. The total alkaloid content of in vitro microtubers elicited with 100 μM SA was equivalent to that of field-grown tubers, while those not treated with SA contained lower alkaloid content. The cytotoxicity assay showed preliminary cytotoxic effects of SA-treated microtubers against the breast cancer cell line SUM159, comparable to field-grown tubers. Transcriptomic analysis revealed many differentially expressed genes (DEGs) in SA-treated in vitro microtubers. Six and twelve DEGs were annotated to the tropane, piperidine, and pyridine alkaloid pathway and the isoquinoline alkaloid pathway, respectively. RT-qPCR confirmed that the genes encoding spermidine synthase (c64642_g1), hyoscyamine (6S)-dioxygenase (c62620_g1), catechol oxidase (c61704_g3), monoamine oxidase (c65996_g3), and aspartate transaminase (c71069_g1) were significantly induced by SA. This study advances the production of P. ternata microtubers without field cultivation and provides considerable genetic information regarding SA-promoted alkaloid accumulation in P. ternata.