In dairy cattle, mammary gland involution serves to remodel the secretory tissue and occurs in a period of overlap between mammogenic stimulation caused by the next developing pregnancy and tissue regression induced by milk stasis. At this time, high concentrations of 17β-oestradiol (E2) and progesterone (P4) support the regeneration of the mammary tissue, as well as enhance autophagy, a cellular process induced in response to stressful conditions for energy generation and homeostasis maintenance. This study aimed to elucidate the mechanisms of autophagy induction by E2 and P4 using an in vitro model of involution based on 20-fold reduction of FBS content (from 10% to 0.5%) in the culture medium of BME-UV1 bovine mammary epithelial cells (MECs). Real-time RT-PCR, Western blot and EMSA analyses demonstrated that addition of E2 and P4 caused a genomic effect in BME-UV1 cells, stimulating the expression of autophagy-related genes (ATGs): BECN1, ATG5, LC3B and their corresponding proteins. Furthermore, knockdown of oestrogen receptor (ERα) and experiments with the use of oestrogen and progesterone antagonists (4-hydroxytamoxifen and RU-486, respectively) demonstrated that the observed genomic effect is mediated by steroid receptors. Finally, both steroids were shown to form complexes with beclin1 and regulate Bcl-2 phosphorylation, indicating that an indirect, non-genomic effect of E2 and P4 may also contribute to autophagy induction in bovine MECs.