Poly(ethylene glycol) (PEG) conjugates of Dicer-substrate small interfering RNA (DsiRNA) have been prepared to investigate a new siRNA release strategy. 3′-sense or 5′-antisense thiol-modified, blunt-ended DsiRNAs, inhibiting enhanced green fluorescent protein (eGFP) expression, were covalently conjugated to PEG with varying molecular weights (2, 10, and 20 kg/mol) through a stable thioether bond using a Michael addition reaction. The DsiRNA conjugates with 2 kg/mol PEG (both 3′-sense or 5′-antisense strand conjugated) and the 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA were efficiently cleaved by recombinant human Dicer to 21-mer siRNA, as determined by gel electrophoresis. Importantly, 2 and 10 kg/mol PEG conjugated to the 3′-sense strand of DsiRNA showed potent gene silencing activity in human neuroblastoma (SH-EP) cells, stably expressing eGFP, at both the mRNA and protein levels. Moreover, the 10 kg/mol PEG conjugates of the 3′-sense strand of DsiRNA were less immunogenic when compared with the unmodified DsiRNA, determined via an immune stimulation assay on human peripheral blood mononuclear cells.