Polyamine-modified diosgenin conjugates were designed and synthesized by two distinct conjugation strategies, which endowed the diosgenin (Dio) with hydrophilic amine units and achieved moderate to excellent yields. In vitro antitumor activities of the corresponding polyamine-diosgenin conjugates (PDCs) were evaluated against six different human cancer cell lines using an MTT assay. The results showed that the PDCs derived from diosgenin exhibited significantly enhanced anticancer activity compared to the parent compound. The conjugates 7, 11, and 13 demonstrated superior cytotoxicity than diosgenin across all tested cell lines, with conjugate 7 displaying the most potent antitumor effect. These findings suggest that the introduction of hydrophilic polyamine moieties into steroidal structure of diosgenin is a promising strategy for developing new anticancer agents.