We previously reported that phospholipase C-d1 (PLC-d1) accumulates in the nucleus at the G1/S transition, which is largely dependent on its binding to phosphatidylinositol 4,5-bisphosphate (1). Here, using small interfering RNA (siRNA) that specifically target rat PLC-d1, we investigated whether this enzyme plays a role in cell cycle control. Inhibiting expression of PLC-d1 significantly decreased proliferation of rat C6 glioma cells and altered S phase progression. [3H]-thymidine labeling and FACS analysis indicated that the rates of G1/S transition and DNA synthesis were enhanced. On the other hand, knockdown cultures released from the G1/S boundary were slower to reach full G2/M DNA content, consistent with a delay in S phase. The levels of cyclin E, a key regulator of the G1/S transition and DNA synthesis, were elevated in asynchronous cultures as well as those blocked at the G1/S boundary. Epifluorescence imaging showed that transient expression of human phospholipase C-d1, resistant to these siRNA, suppressed expression of cyclin E at the G1/S boundary despite treatment of cultures with rat-specific siRNA. While whole cell levels of phosphatidylinositol 4,5-bisphosphate were unchanged, suppression of PLC-d1 led to a significant rise in the nuclear levels of this phospholipid at the G1/S boundary. These results support a role for PLC-d1 and nuclear phospholipid metabolism in regulating cell cycle progression,.