Comprehensive analysis of gene function requires the detailed examination of mutant alleles. In Arabidopsis thaliana, large collections of sequence-indexed insertion and chemical mutants provide potential loss-of-function alleles for most annotated genes. However, limitations for phenotypic analysis include gametophytic or early sporophytic lethality, and the ability to recombine mutant alleles in closely linked genes, especially those present as tandem duplications. Transgene-mediated gene silencing can overcome some of these shortcomings through tissue-specific, inducible and partial gene inactivation, or simultaneous targeting of several, sequence-related genes. In addition, gene silencing is a convenient approach in species or varieties for which exhaustive mutant collections are not yet available. Typically, gene function is reduced post-transcriptionally, effected by small RNAs that act in a sequence-specific manner by base pairing to complementary mRNA molecules. A recently introduced approach is the use of artificial microRNAs (amiRNAs). Here, we review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.