Small RNAs targeted to gene promoters in human cells can mediate transcriptional gene silencing (TGS) by directing silent state epigenetic modifications to targeted loci. Many mechanistic details of this process remain poorly defined, and the ability to stably modulate gene expression in this manner has not been explored. Here we describe the mechanisms of establishment and maintenance of long-term transcriptional silencing of the human ubiquitin C gene (UbC). Sustained targeting of the UbC promoter with a small RNA for a minimum of 3 days resulted in long-term silencing which correlated with an early increase in histone methylation and a later increase in DNA methylation at the targeted locus. Transcriptional silencing of UbC required the presence of a promoter-associated RNA. The establishment and maintenance of the TGS were shown to require distinct protein factors. Argonaute 1 (Ago1), DNA methyltransferase 3a (DNMT3a) and histone deacetylase 1 (HDAC1) were required for the initiation of silencing, and DNA methyltransferase 1 (DNMT1) was necessary for maintenance. Taken together the data presented here highlight the cellular pathway with which noncoding RNAs interact to epigenetically regulate gene expression in human cells.