Small interfering RNAs (siRNAs) are invaluable research tools for studying gene functions in mammalian cells. siRNAs are mainly produced by chemical synthesis or by enzymatic digestion of double-stranded RNA (dsRNA) produced in vitro. Recently, bacterial cells, engineered with ectopic plant viral siRNA binding protein p19, have enabled the production of "recombinant" siRNAs (pro-siRNAs). Here, we describe an optimized methodology for the production ofmilligramamount of highly potent recombinant pro-siRNAs from Escherichia coli cells. We first optimized bacterial culture medium and tested new designs of pro-siRNA production plasmid. Through the exploration of multiple pro-siRNA related factors, including the expression of p19 protein, (dsRNA) generation method, and the level of RNase III, we developed an optimal pro-siRNA production plasmid. Together with a high-cell density fed-batch fermentation method in a bioreactor, we have achieved a yield of similar to 10 mg purified pro-siRNA per liter of bacterial culture. The pro-siRNAs produced by the optimized method can achieve high efficiency of gene silencing when used at low nanomolar concentrations. This new method enables fast, economical, and renewable production of pure and highly potent bioengineered pro-siRNAs at the milligram level. Our study also provides important insights into the strategies for optimizing the production of RNA products in bacteria, which is an under-explored field.