Potent anti-cancer compounds FR901464 and its methyl-ketal derivative spliceostatin A (SSA) inhibit cell cycle progression at G1 and G2/M phases. These compounds bind to the spliceosome and inhibit the splicing reaction. However, the molecular mechanism underlying G1 arrest after SSA treatment remains unknown. In this study, we found that similar to 90% of SSA-treated cells arrested at G1 phase after cell cycle synchronization. SSA treatment caused upregulation of the p27 cyclin-dependent kinase inhibitor both at mRNA and protein levels. In addition to p27, we observed expression of p27*, a C-terminal truncated form of p27 that is translated from CDKN1B (p27) pre-mRNA accumulated after splicing inhibition. Overexpression of p27 or p27* inhibited the exit from G1 phase after a double thymidine block. Conversely, knocking down of p27 by siRNA partially suppressed the G1 phase arrest caused by SSA treatment. There results suggest that G1 arrest in SSA-treated cells is caused, at least in part, by upregulation of p27 and p27*.