Background <p>The study aimed to assess the effects of miR-101-3p on the migratory and invasive abilities of ovarian cancer cells, specifically in the SKOV3 and OVCAR-3 cell lines.</p> Methods <p>The expression of miR-101-3p was overexpressed in SKOV3 and OVCAR-3 ovarian cancer cell lines via mimic transfection. The effects on cell viability, migration, and invasion were evaluated using CCK8, wound healing, and Transwell assays, respectively. RNA sequencing was subsequently performed to identify differentially expressed genes.</p> Results <p>Overexpression of miR-101-3p significantly decreased cell viability, migration, and invasion in both SKOV3 and OVCAR-3 cells. Transcriptomic analysis identified numerous differentially expressed genes, including FN-1 and CXCL8. Intriguingly, while miR-101-3p suppressed FN-1 mRNA in SKOV3 cells, it led to an increase in both FN-1 and CXCL8 mRNA levels in OVCAR-3 cells, despite inhibiting their invasive phenotype.</p> Conclusions <p>Our study confirms that miR-101-3p acts as a tumor suppressor in ovarian cancer. The paradoxical regulation of downstream gene transcripts highlights a complex, cell-type-specific regulatory network. This suggests that the overall phenotypic effect of miR-101-3p is an integrated outcome of modulating multiple targets, rather than a linear pathway, and underscores its potential as a therapeutic target requiring further mechanistic study.</p> Clinical trial number <p>Not applicable.</p>