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Staphylococcus saprophyticus (S. saprophyticus) is one of the most common bacteria that cause significant illness owing to the development of antibiotic resistance. Antibiotic-resistant microorganisms, such as S. saprophyticus, need novel research and development approaches. Small interfering RNA (siRNA) duplexes of 21-23 bp were synthesized against the mRNA of the msrA gene in S. saprophyticus to evaluate the effect of siRNA in the msrA gene expression. Reverse transcription PCR (RT-PCR) was used for detection the effect of the siRNA in msrA mRNA expression. The disc diffusion technique was used to assess differences of the erythromycin susceptibility in response to the action of siRNA. In vitro, msrA-siRNAs reduced both mRNA expression and activity of S. saprophyticus. The effectiveness of siRNA was assessed by comparing the antibacterial activity of S. saprophyticus before and after the introduction of msrA-siRNA to the bacterium. Two msrA-siRNA sequences, siRNA1 and siRNA2 were used in study. qRT-PCR revealed that two msrA-siRNA sequences strongly suppressed the production of msrA-mRNA, P = (0.010, and 0.002 ) respectively at (P< 0.05) when in comparison with control and P = (0.015, and 0.028 ), when compared to scrambled-siRNA, respectively. Regarding the effect of treatment the isolates with siRNA, there were 5/9 (55.5%) of resistant isolates, sensitive to erythromycin. According to the findings, siRNA targeting of msrA looks to be a novel strategy for treating S. saprophyticus illnesses.