Endogenous siRNAs (endo-siRNAs) are key regulators of RNA silencing in plants and worms; however, the biogenesis and function of endogenous siRNAs in mammals remain largely unknown. We previously demonstrated that human telomerase reverse transcriptase produces a self-targeting endogenous siRNA from non-coding RMRP RNA via RNA-dependent RNA polymerase (RdRP) activity. Here, we investigated whether the endo-siRNA derived from RMRP targets other genes in addition to RMRP. Four algorithms for microRNA target prediction were used to identify possible targets of the endo-siRNA, and the phytanoyl-CoA hydroxylase-interacting protein-like gene (PHYHIPL) was identified as the most promising candidate. The 3' UTR of PHYHIPL was found to contain three possible target sites with perfect seed pairing; deletion of each of these sites resulted in recovery of upstream luciferase expression. In addition, sequence-specific inhibition of the RMRP-derived endo-siRNA increased expression of PHYHIPL mRNA. The results described here suggest that the endo-siRNA uses silencing mechanisms that are similar to those used by microRNAs for gene silencing. To our knowledge, this study is the first confirmation of the off-target effect of human endogenous siRNA produced by RdRP activity.