Pituitary adenomas account for ~10% of intracranial tumors and result in morbidity due to both altered hormonal patterns as well as side effects of therapy. Currently, great attention has been given to preventive strategies, and in this context, the use of functional foods with chemopreventive compounds appears to contribute much in this process. Among these substances, we highlight the consumption of carotenoids is associated with reduced risk of chronic diseases including cancer and vascular diseases. This study aimed to evaluate the influence of β-carotene and lycopene in proliferation, cell cycle and apoptosis of mouse ACTH-secreting pituitary adenoma cells (AtT-20). AtT-20 cells were incubated with DMEM supplemented with 10% fetal bovine serum under an atmosphere of 5% CO2 at 37 °C. The cells were incubated with different concentrations of carotenoids (0.540 μM) for 48 and 96 h. Assessment of cell proliferation was assessed using the colorimetric MTT. The analysis by flow cytometry was used to assess the distribution of the phases of the cell cycle. Apoptosis was assessed using the annexin V-fluorescein isothiocyanate apoptosis detection kit I. The results of cell viability showed that both lycopene and β-carotene decreased the proliferation of AtT-20 after 48 and 96 h. The cell cycle analysis revealed that lycopene increased proportion of cell line AtT-20 in the G0/G1 phase and decreased cells in S and G2/M phases after 96 h. Furthemore, β-carotene induced an accumulation in S and G2/M phases in the line AtT-20 after 96 h. The process of apoptosis was induced by β-carotene (10 μM) after 96 h, and lycopene in a dose-dependent manner induced apoptosis after 48 and 96 h. Lycopene and β-carotene showed as potent inhibitors of cell growth, suggesting a modulation of the cell cycle of pituitary tumor cells, with an increase in apoptosis and the effect was dose and time dependent.