BackgroundISGylation is a post-translational protein modification that regulates many life activities, including immunomodulation, antiviral responses, and embryo implantation. The exact contribution of ISGylation to folliculogenesis remains largely undefined.ResultsHere,Isg15knockout in mice causes hyperfertility along with sensitive ovarian responses to gonadotropin, such as increases in cumulus expansion and ovulation rate. Moreover, ISG15 represses the expression of ovulation-related genes in an ISGylation-dependent manner. Mechanistically, ISG15 binds to ADAMTS1 via the ISG15-conjugating system (UBA7, UBE2L6, and HERC6), ISGylating ADAMTS1 at the binding sites Lys309, Lys593, Lys597, and Lys602, resulting in ADAMTS1 degradation via a 20S proteasome-dependent pathway.ConclusionTaken together, the present study demonstrates that covalent ISG15 conjugation produces a novel regulatory axis of ISG15-ADAMTS1 that enhances the degradation of ADAMTS1, thereby compromising ovulation and female fertility.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-023-01024-4.