A new labdane-type diterpenoid, ent-19-ol-13-epi-manoyl oxide,19-undecane ester, together with ten known diterpenes, were isolated from the ethanolic crude extract of the fresh tubers of Sagittaria trifolia L. The chemical structures of these compounds were determined by extensive 2-D NMR experiments and by comparison with the data reported in the literature. These compounds showed different inhibitory effects on various human cancer cells. Among these, compound 11 exhibited potential inhibition effects against human colon cancer cells. Moreover, flow cytometry demonstrated that compound 11 arrested the cell cycle at the G1 phase and induced cellular apoptosis, accompanied by mitochondrial membrane potential reduction. Mechanistic studies revealed that treatment with compound 11 inhibited IKKα/β phosphorylation and IκBα phosphorylation, which subsequently caused the blockage of NF-κB p65 phosphorylation and nuclear translocation. Compound 11 also inhibited the expression of c-Myc, Cyclin D1, and Bcl-2, the downstream targets of NF-κB. Therefore, our findings provided insight into the anticancer components of Sagittaria trifolia L. tubers, which could facilitate their utilization as functional food ingredients.