Giardia lamblia is one of the most common parasites of humans and a major cause of diarrhea worldwide. Giardia undergoes antigenic variation. Only one variant-specific surface protein (VSP), from a repertoire of approximately 150 vsp genes, is expressed on the surface of every trophozoite, but switching to the expression of distinct VSPs occurs even in absence of immune pressure. Here we show that vsp expression is regulated by a mechanism displaying features of RNAi. Giardia clones expressing a single surface antigen efficiently transcribe several other vsps but only accumulate transcripts encoding the actual VSP. Detection of antisense RNAs corresponding to the silenced vsps likely generated by a cellular RNA-dependent RNA-polymerase, ATP-dependent cleavage of double stranded RNAs into small RNA fragments from the silenced but not for the expressed vsp, and characterization of post-transcriptional gene silencing (PTGS) components indicate that Giardia possesses active Dicer and RISC systems. Moreover, knock-down of these enzymes leads to a change from single to multiple VSP expression in individual trophozoites, demonstrating the involvement of a PTGS mechanism in controlling antigenic variation of this important human pathogen.