Plumbagin (1), a naphthoquinone, induces cell death and affects various signaling pathways in cancer cells. Wnt signaling is active constitutively in colorectal cancer and plays an important role in its progression and pathogenesis. It was hypothesized that 1 is likely to modulate Wnt signaling, and this compound was studied for its effect on this pathway in human colorectal cancer cells. Plumbagin (1) was found to downregulate Wnt signaling when assessed by a TOPFlash/FOPFlash reporter activity assay and also decreased the expression of several coactivators and downstream targets of Wnt signaling such as β-catenin, TCF7L2, p300, Bcl9l, c-Myc, vimentin, and cyclinD1 in SW620 colorectal cancer cells. Using isogenic HCT116p53+/+ and HCT116p53-/- colorectal cancer cells, it was found that compound 1-mediated downregulation of Wnt signaling is p53-independent. Interestingly, treatment with 1 upregulated the expression of HBP1 (a negative regulator of Wnt signaling) in these cells. The results obtained show for the first time that downregulation of Wnt signaling could be one of the molecular mechanisms by which plumbagin exerts its inhibitory effects in human colorectal cancer cells.