Phospholipase Cβ1 (PLCβ1) is a G-protein-regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have previously found that in solution PLCβ1 binds to the RNA processing protein translin-associated factor X (TRAX) with nanomolar affinity and that this binding competes with G proteins. Here, we show that endogenous PLCβ1 and TRAX interact in SK-N-SH cells and also in HEK293 cells induced to overexpress PLCβ1. In HEK293 cells, TRAX overexpression ablates Ca(2+) signals generated by G protein-PLCβ1 activation. TRAX plays a key role in down-regulation of proteins by small, interfering RNA, and PLCβ1 overexpression completely reverses the 2- to 4-fold down-regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by an ∼4-fold recovery in both the transcript and protein levels. Also, down-regulation of endogenous PLCβ1 in HEK293 and HeLa cells allows for an ∼20% increase in siRNA(GAPDH) silencing. While PLCβ1 overexpression results in a 50% reversal of cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g., cyclophilin, Hsp90, translin). PLCβ1 overexpression in HEK293 and HeLa cells causes a 30% reduction in the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis that allows cells to progress through the S phase. We find that PLCβ1 reverses the cell death and completely rescues H2B levels caused by siRNA knockdown of LDH or GAPDH. Taken together, our study shows a novel role of PLCβ1 in gene regulation through TRAX association.