Background: There are a limited number of studies investigating the side effects and effectiveness of various doses of isotretinoin (ISO). We have previously shown that high-dose ISO affects pituitary hormones. Objectives: To our knowledge, there is no study in the literature looking into the effects of various doses of ISO on pituitary hormones. We searched pituitary hormones in three groups of different doses in acne patients. Methods: We included 105 acne vulgaris patients from two different centers. We divided the patients into three groups; the first group received 0.5-1 mg/kg/day, the second 0.2-0.5 mg/kg/day and the third intermittent 0.5-1 mg/kg/day (only 1 week in 1 month) ISO treatment. Blood samples were collected for biochemistry and hormone analysis, before the treatment and after 3 months. Results: After 3 months of treatment with ISO, luteinizing hormone (LH) (p < 0.001), prolactin (p < 0.001), total testosterone (p < 0.001), adrenocorticotropic hormone (ACTH) (p < 0.001), cortisol (p < 0.001), insulin-like growth factor-binding protein 3 (p < 0.001), insulin-like growth factor 1 (IGF-1) (p = 0.002), growth hormone (GH) (p = 0.002) and free T3 (fT3) (p < 0.001) levels had decreased significantly. Furthermore, we split data into three different groups. Among the patients receiving intermittent-dose ISO, LH, ACTH, IGF-1, GH and fT3 measurements lost significance. Most of the significant measurements observed in the whole group were also significant among the patients receiving high-dose ISO. Additionally, dehydroepiandrosterone sulfate (p = 0.003) levels increased, and free T4 levels decreased significantly. Conclusions: ISO affects pituitary hormones at all of these three doses. The differences in pituitary hormones are more pronounced in high-dose treatment. The weakest effect was observed in the intermittent-dose group. Choosing lower doses of ISO may decrease side effects, however the effectiveness of the treatment may also be diminished. ISO, by affecting the PPARγ/RXR system, may affecting hormone systems. These changes in various hormone systems may be related to the effectiveness of ISO.