Objectives: Epidemiological observations indicate that female flight attendants are exposed to some reproductive and endocrine system disturbances. The aim of the study was to determine the incidence of hyperprolactinemia among female flight attendants, and to identify factors affecting the secretion of prolactin in female flight attendants working within 1 time zone as well as on long-distance flights. Material and Methods: The cross-sectional study covered 103 women aged 23–46 years. The study group (I) was divided into 2 subgroups: subgroup Ia comprising female flight attendants flying within 1 flight zone, and subgroup Ib composed of female flight attendants working on long-distance flights. The control group (II) included women of reproductive age who sought medical assistance due to marital infertility in whom the male factor was found to be responsible for problems with conception in the course of the diagnostic process. The assessment included: age, the body mass index, menstrual cycle regularity, the length of service, the frequency of flying, the prolactin, estradiol and progesterone concentrations, and the result of endometrial biopsy. Descriptive and inferential statistics methods were used to compile the data. Results: The incidence of hyperprolactinemia in the female flight attendants (46%) was significantly higher than in the control group (9%), p < 0.001. Differences between subgroups Ia and Ib regarding individual concentrations were not statistically significant (p = 0.425). Hyperprolactinemia among the female flight attendants working ≥15 years is present slightly more often than in those working <15 years: 46% vs. 45% (p > 0.05). No significant difference was revealed in the secretion of prolactin between the study participants spending <60 h/month flying and those spending ≥60 h/month flying (p > 0.05). Conclusions: Hyperprolactinemia is more common in female flight attendants than in the general population. High values of prolactin concentration in flight attendants are rarely manifested in clinical symptoms. The frequency of flying and the length of service do not affect the development of hyperprolactinemia or the mean prolactin concentration. Int J Occup Med Environ Health. 2021;34(3):351–61