Background: Natural products with unique mechanisms remain of great interest because targeted cancer therapies frequently fail due to toxicity or resistance. Cardiac glycosides have demonstrated antitumor activity, but whether their effects involve microRNA regulation remains largely unexplored. This study investigates whether ouabain derived from Strophanthus gratus (Wall. & Hook. ex Benth.) Baill. (SGO) exerts its antitumor effects through miR-145-5p, a known tumor suppressor, using both colorectal and breast cancer models. Methods: We performed transcriptomic profiling in HCT116 colorectal cancer cells treated with SGO, followed by in vitro assays—including cell viability, caspase 3/7 activity, flow cytometry, and colony formation—in HCT116 and MCF-7 breast cancer cells. In vivo efficacy was evaluated using HCT116 xenograft models in BALB/c-nu/nu mice. miR-145-5p gain- and loss-of-function approaches were employed to determine its functional requirement. Results: SGO dose-dependently suppressed proliferation, induced apoptosis, and inhibited colony formation in both colorectal (HCT116) and breast (MCF-7) cancer cells, and significantly upregulated miR-145-5p levels in both cell types. Transcriptomic analysis identified miR-145-5p as a highly differentially expressed miRNA. In HCT116 xenograft models, SGO inhibited tumor growth by approximately 60% and elevated intratumoral miR-145-5p levels. Importantly, inhibition of miR-145-5p significantly attenuated these effects both in vitro and in vivo, establishing that the antitumor activity of SGO depends on the upregulation/activation of miR-145-5p in both cancer types. Conclusions: We have found that SGO inhibits colorectal and breast cancer growth through a miR-145-5p-dependent mechanism, revealing a previously unrecognized regulatory axis for cardiac glycosides. These findings position SGO as a promising candidate for further preclinical studies and suggest that pharmacologic re-expression of miR-145-5p may represent a viable therapeutic strategy in targeted therapy.