BACKGROUND: Studies have indicated that X-linked microRNAs (miRNAs) play a role in the pathogenesis of rheumatoid arthritis (RA) and its gender-specific differences. However, research on specific miRNAs remains limited. This study aims to investigate the possible role of X-linked miR-542-5p in RA pathogenesis and gender differences. METHODS: We investigated the impact of miR-542-5p on RA pathogenesis and gender differences by manipulating its expression in various rat models. RESULTS: Our findings revealed a significant overexpression of miR-542-5p in RA patients compared with healthy individuals, with a notable gender difference among RA patients. In vivo experiments confirmed that upregulation of miR-542-5p could accelerate RA pathogenesis. Further analysis showed that the onset of adjuvant-induced arthritis (AIA) in rats exhibited significant gender differences, with more severe clinical phenotypes found in female rats. This may be attributed to their stronger immune responses and elevated levels of miR-542-5p. Subsequent in vitro and in vivo experiments demonstrated that miR-542-5p contributes to the regulation of gender differences in RA pathogenesis by promoting the differentiation of Th17 cells. CONCLUSIONS: This study offers new insights into the sex-specific nature of RA, suggesting X-linked miR-542-5p as a potential target for both diagnostic and therapeutic purposes. These findings lay the groundwork for the development of gender-specific therapeutic strategies for RA and underscore the importance of gender consideration in RA research.