We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 weeks, using a whole body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of pro-autophagic genes Cdkn1b, Map1lc3a, Bad and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3 and Maplc31b and a significant decrease in Akt1 and Mtor expression was detected by q-PCR. AMPK alpha1+alpha2 and ATG7 protein expression was significantly increased whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed vs. control ovaries. Up-regulation of AMPK alpha1+alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two pro-survival factors AKT and mTOR were decreased in expression, an outcome that favors induction of the autophagy pathway whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of anti-autophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.