Abstract: The clinical utility of macrolide antibiotics has declined due to the appearance of resistant isolates. A spiramycin I-resistant Staphylococcus aureus ATCC29213-R was induced and isolated with increasing the concentration of spiramycin I, which exhibits an A→C transversion at position 2089 in the 23S rRNA gene, which is first reported in the S. aureus. A RNA-seq based transcriptomic analysis was performed to understand the overall response of resistant bacteria to spiramycin I treatment with subinhibitory dosage. In this study, There are a total of 322 up-regulated and 82 down-regulated genes in spiramycin I-treated S. aureus ATCC29213-R and 426 up-regulated, 838 down-regulated in spiramycin I-treated S. aureus ATCC29213, which were identified differentially expressed compared to their control with a minimum 2-fold change(Q< 0. 05). Interestingly, The data showed that argH and argG transcripts, in the arginine biosynthetic pathway, were decreased by 13. 51-fold and 21. 45-fold, respectively, compared to the control, while the expression level of three genes involved in arginine catabolism, arcA, arcC, and argF, increased by 35-fold, 18. 05-fold and 30. 84-fold, respectively. The results revealed that spiramycin I could trigger the up-regulation of the genes of ACME-Arc system which allows S. aureus to survive in acidic environments of human skin. This suggesed the arginine-deiminase pathway may be a potential target for treatment of the resistant S. aureus.