Cardiac glycosides (CGs) are natural compounds used to treat congestive heart failure. They have garnered attention as a potential cancer treatment option, especially because they bind to Na⁺/K⁺-ATPase as a target and activate intracellular signaling pathways leading to a variety of cellular responses. In this study we evaluated AMANTADIG, a semisynthetic cardenolide derivative, for its cytotoxic activity in two human androgen-insensitive prostate carcinoma cell lines, and the potential synergistic effects with docetaxel. AMANTADIG induced cytotoxic effects in both cell lines, and a combination with docetaxel showed a moderate and strong synergism in DU145 and PC-3 cells, respectively, at concentrations considerably lower than their IC₅₀ values. Cell cycle analyses showed that AMANTADIG and its synergistic combination induced G2/M arrest of DU145 and PC-3 cells by modulating Cyclin B1, CDK1, p21 and, mainly, survivin expression, a promising target in cancer therapy. Furthermore, AMANTADIG presented reduced toxicity toward non-cancerous cell type (PBMC), and computational docking studies disclosed high-affinity binding to the Na⁺/K⁺-ATPase α subunit, a result that was experimentally confirmed by Na⁺/K⁺-ATPase inhibition assays. Hence, AMANTADIG inhibited Na⁺/K⁺-ATPase activity in PC-3 cells, as well as in purified pig kidney at nanomolar range. Altogether, these data highlight the potent effects of AMANTADIG in combination with docetaxel and offer important insights for the development of more effective and selective therapies against prostate cancer.