RNA interference (RNAi) is induced by the direct transfer of double-stranded RNAs (dsRNAs) into protoplasts prepared from Arabidopsis thaliana seedlings. In this protoplast RNAi system, we compared the efficacies of various-sized dsRNAs (between 21 and 139 nucleotides [nt]) for inducing RNAi and assessed the dsRNA-cleaving activities of Dicer-like 3 (DCL3) and 4 (DCL4). After the direct transfer of dsRNAs into protoplasts, cleaved RNA products of 21 nt were detected from long 130- or 500-nt dsRNAs by DCL4 but not from 37-nt dsRNAs. These results indicate that DCL4 preferentially cleaves long dsRNAs in protoplasts, consistent with our previous biochemical data regarding the substrate specificity of DCL4. Direct transfer of long dsRNAs of approximately 130 nt into protoplasts induces RNAi much more effectively (by approximately 60- to 400-fold) than direct transfer of short 37-nt dsRNAs. Although transfer of 21-nt dsRNAs into protoplasts induced RNAi without DCL4 activity, the induction of RNAi was less effective (by approximately 0.01-fold) compared with long dsRNAs. These results indicate that cleavage of long dsRNAs exceeding 100 nt by DCL4 into 21-nt dsRNAs is essential for efficient induction of RNAi in plant cells.