Background <p>Recurrent spontaneous abortion (RSA) affects approximately 1–5% of childbearing women and poses a significant threat to global reproductive health. Transfer RNA-derived small RNAs (tsRNAs) are a novel class of noncoding RNAs implicated in various human diseases. However, the role and mechanism of tsRNAs in regulating trophoblast function during RSA development remain unknown.</p> Methods <p>High-throughput sequencing was performed to analyze the differential tsRNAs in the villous tissues of patients with RSA and controls. CCK-8, transwell assay, and flow cytometry were performed to detect the effects of tRF-5028c on proliferation, migration, invasion, and apoptosis of human extravillous trophoblast cell line HTR-8/SVneo. The target genes of tRF-5028c were predicted via bioinformatic analysis and verified by dual luciferase reporter gene assay. Moreover, pregnant mice were injected with tRF-5028c mimics to confirm the findings in vivo.</p> Results <p>A total of 1907 tsRNAs were detected, of which 298 were differentially expressed in the villous tissues. tRF-5028c was significantly upregulated in the RSA group compared with control. Functionally, tRF-5028c overexpression inhibited HTR-8/SVneo cell proliferation, migration, and invasion and promoted apoptosis, whereas tRF-5028c knockdown showed opposite effects. Mechanically, tRF-5028c suppressed CRKL expression by directly binding to its 3′-untranslated region, thus inactivating the downstream C3G/Rap1 signaling pathway. Finally, tRF-5028c mimics injection increased embryo absorption rate in mice.</p> Conclusions <p>tRF-5028c upregulation impaired trophoblast function to facilitate RSA development by directly targeting CRKL-mediated Rap1 pathway. The findings provide the first evidence of tsRNA dysregulation in RSA pathogenesis and lay a foundation for potential targeted therapies.</p>