Anomanolide C (AC), a natural withanolide isolated fromTubocapsicum anomalum, has been reported to have exhibits remarkable anti-tumour activities in several types of human cancers, particularly triple-negative breast cancer (TNBC). However, its intricate mechanisms still remain need to be clarified. Here, we evaluated whether AC could inhibit cell proliferation and the role of AC in ferroptosis induction and autophagy activation. Subsequently, the anti-migration potential of AC was found via autophagy-dependent ferroptosis.Additionally, we found that AC reduced the expression of GPX4 by ubiquitination and inhibited TNBC proliferation and metastasisin vitroandin vivo.Moreover, we demonstrated that AC induced autophagy-dependent ferroptosis, and led to Fe2+accumulationviaubiquitinating GPX4. Moreover, AC was shown to induce autophagy-dependent ferroptosis as well as to inhibit TNBC proliferation and migrationviaGPX4 ubiquitination. Together, these results demonstrated that AC inhibited the progression and metastasis of TNBC by inducing autophagy-dependent ferroptosisviaubiquitinating GPX4, which might shed light on exploiting AC as a new drug candidate for the future TNBC therapy.