Artificial microRNAs (amiRNAs) are highly specific gene silencing tools in Arabidopsis. Unlike animal miRNAs, which often target many genes with limited complementarity, plant miRNAs typically target few genes with high sequence complementarity. This study demonstrates that amiRNAs, designed to target specific genes or groups of genes, exhibit similar specificity to natural plant miRNAs. amiRNAs are effective for silencing multiple related genes and can be expressed under tissue-specific or inducible promoters with limited nonautonomous effects. The design principles of amiRNAs have been integrated into a web-based tool for automated design. The study shows that amiRNAs can efficiently silence target genes, with little evidence of secondary siRNA formation, consistent with natural plant miRNAs. Known parameters for target selection by natural miRNAs accurately predict direct targets of amiRNAs, indicating that the plant silencing machinery acts with high specificity. amiRNAs are also effective when expressed under inducible promoters and have limited nonautonomous effects. The study also demonstrates that amiRNAs can target multiple genes, including those involved in flowering time and floral patterning. For example, amiR-trichome targets three MYB genes involved in trichome patterning, while amiR-mads-1 and amiR-mads-2 target MADS box genes involved in flowering time and floral patterning. amiR-yabby-1 and amiR-yabby-2 target YABBY genes involved in leaf polarity. The specificity of amiRNAs was confirmed through microarray analysis, showing that significantly downregulated genes were not more similar to the respective amiRNAs than all genes. This suggests that amiRNAs are as specific as natural miRNAs. Additionally, the study shows that amiRNAs can be used for spatially and temporally restricted gene silencing, with effects that are not transmitted autonomously. The study also highlights the advantages of amiRNAs over conventional RNAi, including their ability to target specific genes and their potential for allele-specific knockouts. amiRNAs are particularly useful for targeting groups of closely related genes, including tandemly arrayed genes. The study concludes that amiRNAs are a powerful tool for gene silencing in plants, with high specificity and minimal off-target effects.Artificial microRNAs (amiRNAs) are highly specific gene silencing tools in Arabidopsis. Unlike animal miRNAs, which often target many genes with limited complementarity, plant miRNAs typically target few genes with high sequence complementarity. This study demonstrates that amiRNAs, designed to target specific genes or groups of genes, exhibit similar specificity to natural plant miRNAs. amiRNAs are effective for silencing multiple related genes and can be expressed under tissue-specific or inducible promoters with limited nonautonomous effects. The design principles of amiRNAs have been integrated into a web-based tool for automated design. The study shows that amiRNAs can efficiently silence target genes, with little evidence of secondary siRNA formation, consistent with natural plant miRNAs. Known parameters for target selection by natural miRNAs accurately predict direct targets of amiRNAs, indicating that the plant silencing machinery acts with high specificity. amiRNAs are also effective when expressed under inducible promoters and have limited nonautonomous effects. The study also demonstrates that amiRNAs can target multiple genes, including those involved in flowering time and floral patterning. For example, amiR-trichome targets three MYB genes involved in trichome patterning, while amiR-mads-1 and amiR-mads-2 target MADS box genes involved in flowering time and floral patterning. amiR-yabby-1 and amiR-yabby-2 target YABBY genes involved in leaf polarity. The specificity of amiRNAs was confirmed through microarray analysis, showing that significantly downregulated genes were not more similar to the respective amiRNAs than all genes. This suggests that amiRNAs are as specific as natural miRNAs. Additionally, the study shows that amiRNAs can be used for spatially and temporally restricted gene silencing, with effects that are not transmitted autonomously. The study also highlights the advantages of amiRNAs over conventional RNAi, including their ability to target specific genes and their potential for allele-specific knockouts. amiRNAs are particularly useful for targeting groups of closely related genes, including tandemly arrayed genes. The study concludes that amiRNAs are a powerful tool for gene silencing in plants, with high specificity and minimal off-target effects.