In Trypanosoma brucei, Argonaute1 (TbAGO1) is an essential component of the RNA interference (RNAi) pathway. While characterizing a TbAGO1 conditional knockout cell line, we discovered that, upon blockage of TbAGO1 transcription, the RNAi response to transfected double-stranded RNA was severely inhibited, although there was no change in the TbAGO1 protein level. This observation suggested that steady-state TbAGO1 was not sufficient to fully support the RNAi response to transfected dsRNA and implicated newly synthesized Argonaute in this phenomenon. Indeed, a translational blockade of TbAGO1 mRNA with an antisense morpholino oligonucleotide resulted in inhibition of the RNAi response, even though the steady-state level of TbAGO1 remained unchanged during the time of the assay. Thus, we concluded that in T. brucei, newly synthesized TbAGO1 is required to support an efficient RNAi response. We speculate that newly processed siRNAs may be preferentially loaded onto newly synthesized TbAGO1, and this mechanism may contribute to the homeostasis of the RNAi pathway.