The aim of the study was to determine whether two postpartum intramuscular treatments with 200 mg of beta-(β-)carotene (Carofertin; Alvetra u. Werfft, Vienna, Austria) in a 14-day interval increases β-carotene concentrations in blood, particularly around the time of the first artificial insemination (AI), and to test the effect of the treatment on fertility parameters, luteal size, and progesterone blood levels of dairy cows. A total of 297 Holstein dairy cows were enrolled in the study. Between 28 and 34 days postpartum (dpp) β-carotene concentrations were measured in blood samples using an on-site test (iCheck carotene; BioAnalyt, Teltow, Germany). Cows with a β-carotene concentration <3.5 mg/L, indicating a deficiency of β-carotene, were allocated either to the β-carotene treatment group BCT (n = 123) or to the control group CON (n = 121). Cows with concentrations ≥3.5 mg/L were assigned to an optimally supplied reference group (REF; n = 53). Cows in the BCT group received 200 mg of β-carotene intramuscularly at 28–34 dpp and at 42–48 dpp. Further blood samples were collected at 35–41 dpp, 42–48 dpp, 49–55 dpp, and in the week after the first AI and their β-carotene concentrations were analyzed. Between day 10 and 14 after the first AI, the blood progesterone concentration was measured and the size of the corpus luteum (CL) was determined by ultrasound. Blood β-carotene concentrations increased in the BCT cows in the week after the treatment with a peak at 49–55 dpp and were significantly higher than in the CON group at each time point after the first treatment. Logistic regression models, however, revealed that the treatment with β-carotene had no effect on first service conception rate, days to first service, time to pregnancy, or percentage of pregnant cows within 150 dpp. Furthermore, there was no effect on progesterone concentration or the size of the CL between the groups. In conclusion, two treatments with Carofertin postpartum increased β-carotene blood concentrations but had no effect on the fertility parameters in this study.