Uterine fibroids (UF) are the most common pelvic tumors in women of reproductive-age and they usually cause heavy menstrual bleeding, pain and infertility. Autophagy is a collection of processes that enables the cells to digest and recycle their cytoplasmic contents, such as toxic protein aggregates, defunct or disused organelles and invading microorganisms. Dysregulation in autophagy process were described in neoplasms; however, the contribution of autophagy to the pathogenesis of UF remains unknown. In this study, we demonstrate that autophagy is deregulated in human UF as evidenced by significant accumulation of autophagosome in human UF cells compared to normal myometrium cells. Analysis of the autophagy markers revealed an enhanced initiation of the autophagy in UF tissues compared to their adjacent myometrial tissues (MyoF). However, autophagosome maturation and flux was blocked in UF tissues, as marked by accumulation of LC3-B and P62 protein. This block was associated with defective expression of autophagy-related protein 4 (ATG4) in the UF tissues compared to MyoF in ~90% of patient samples. Silencing of ATG4D in normal human myometrial cells resulted in defective autophagy flux, enhanced cell proliferation and increased extracellular matrix production, which phenocopy UF cell line. This study indicates that impairment of autophagy flux secondary to defective expression of ATG4D expression is a new mechanistic aberration that contributes to UF pathogenesis. Targeting autophagy pathway could provide novel medical therapeutic approach for non-surgical treatment of UF.