Withanolides are generally defined as C(28) steroidal lactones built on an intact or rearranged ergostane skeleton and have been shown to exhibit antiproliferative activity on various types of cancer cells. In this study, we investigated the effect of a new withanolide Tubocapsanolide A isolated from Tubocapsicum anomalum and addressed its molecular action. Tubocapsanolide A inhibited proliferation of A549, H358, and H226 human lung cancer cells via induction of G(1) growth arrest. We found that Tubocapsanolide A treatment led to up-regulation of cyclin E, p21, and p27, whereas other cyclins and cyclin-dependent kinases were not affected in A549 cells. Conversely, Skp2, the F-box protein that is implicated in the mediation of degradation of p21 and p27, was significantly down-regulated. Chromatin immunoprecipitation assay suggested that Tubocapsanolide A suppressed Skp2 expression by inhibiting the binding of Rel A to the nuclear factor-kappaB site of Skp2 gene promoter. In addition, we showed that inhibition of Skp2 is a critical step for the suppression of cell proliferation by Tubocapsanolide A because ectoexpression of Skp2 effectively reversed Tubocapsanolide A-induced p27 up-regulation and growth inhibition in human lung cancer cells. Collectively, we have identified Skp2 as a molecular target for Tubocapsanolide A and suggest that this withanolide may be useful for the prevention or treatment of cancer cells with Skp2 overexpression.