Accumulated evidence suggests that connexin43 (Cx43) serves as a tumor-suppressing gene. We have previously shownA. B. that Cx43 suppressed the G(1)-S phase cell cycle transition via increasing the level of p27 (Zhang, Y. W., et al., Oncogene, 20: 4138-4149, 2001). Here we report that Cx43 inhibited expression of Skp2, the human F-box protein that regulates p27 ubiquitination. This reduction was attributed to an increased degradation of Skp2. The Cx43 antisense oligonucleotide blocked this inhibitory effect of Cx43 on Skp2 expression and led to p27 down-regulation. In contrast, the antisense oligonucleotide of Skp2 induced a further increase in the level of p27. However, ectopic expression of Skp2 reversed the Cx43-induced Skp2 reduction, p27 accumulation, and cell proliferation inhibition. Cx43 increased p27 expression only in the SKP2 +/+ mouse embryo fibroblasts (MEFs), but not in the SKP2 -/- MEFs, indicating that Skp2 plays a critical role in the Cx43-induced p27 up-regulation. We also show that both Skp2 and p27 are required for Cx43 to inhibit cell proliferation, in that Cx43 hardly inhibited cell proliferation of the SKP2 -/- and p27 -/- MEFs, whereas it clearly did both in the SKP2 +/- and in the p27 +/- MEFs. Our findings suggest a new route for Cx43 to inhibit tumor growth by linking it with the key cell cycle regulators.