Cigarette smoking among reproductive-aged women is increasing worldwide. Cigarette smoking is a lifestyle behavior associated with important adverse health effects including subfertility and premature ovarian failure. We previously demonstrated that cigarette smoke (CS) exposure in mice decreases the primordial follicle pool; however, the mechanism of action is largely unknown. Therefore, the present study was designed to elucidate the mechanisms underlying CS exposure-induced ovarian follicle loss. CS exposure induced a significant decrease in the relative ovarian weight and the number of primordial and growing follicles. Oxidative stress, as shown by increased Hsp25 and decreased superoxide dismutase 2 protein expression, was found in mice exposed to CS for 8 weeks. Exposure decreased Bcl-2 but failed to induce apoptosis. An increased number of autophagosomes in granulosa cells of ovarian follicles together with increased expression of Beclin-1 and microtubule-associated protein light chain 3, key regulatory proteins in the autophagy (Atg) pathway, was found in CS-exposed mice compared with the control group. Taken together, our results suggest that CS exposure does not induce apoptosis but rather activates the Atg pathway ultimately leading to ovarian follicle loss. We further postulate that Atg is a novel mechanism of toxicant-induced ovarian follicle loss.