This study was conducted to evaluate if addition of a second prostaglandin (PG) F_2α dose improves fertility of anestrous lactating Holstein cows submitted to an estradiol/progesterone (P4) based timed AI (TAI) protocol. Ovaries were scanned by ultrasound to determine if a corpus luteum (CL) was present at the protocol initiation (d-11) and on the day of PGF_2α (d −4). Cows without CL on d −11 and d −4 were classified as anestrous (n = 436). Anestrous cows were submitted to the TAI protocol: d −11: 2 intravaginal P4 devices (1.9g P4, CIDR, Zoetis) and 2.0 mg of estradiol benzoate (EB) i.m. (Estrogin, Farmavet); d −4, 25mg of PGF2a i.m. (Lutalyse, Zoetis) and withdrawal of one CIDR; d −2 withdrawal of the second CIDR and 1.0 mg i.m. of estradiol cypionate (ECP, Zoetis); on d 0 TAI. On d-4, cows were randomly assigned to 1 of 2 treatments: one dose of PGF_2α on d −4 (1PGF) or 2 doses of PGF_2α (2PGF), the first on d −4 and the second on d −2. Rectal temperature was measured on the day of TAI and 7 d later. The average rectal temperature was calculated and the cows were divided into 2 groups: without hyperthermia (<39.1°C) or with hyperthermia (≥39.1°C). Pregnancy was diagnosed 60 d after AI. Binomial variables were analyzed using the GLIMMIX and continuous using the MIXED procedures of SAS. The 2PGF treatment tended to improve the synchronization rate (80.2% [n = 218] vs. 72.4% [n = 218] P = 0.07). An interaction (P = 0.05) between treatment and hyperthermia was observed for pregnancy per AI (P/AI) on d 60. For hyperthermic cows, P/AI were 9.1% (n = 123) and 9.4% for 1PGF and 2PGF, respectively. For cows without hyperthermia, P/AI were 16.3% (n = 95) and 30% (n = 88) in 1PGF and 2PGF, respectively. When only ovulated cows were included in the analysis, then an interaction (P = 0.05) existed between treatment and hyperthermia on P/AI at 60 d. For cows with hyperthermia, P/AI were 13.8% (n = 80) and 12.1% (n = 100) in 1PGF and 2PGF, respectively. For cows not suffering of hyperthermia, P/AI were 19.4% (n = 78) and 34.8% (n = 75) in 1PGF and 2PGF, respectively. The addition of a second PGF dose during the TAI protocol increased fertility in anestrous dairy cows without hyperthermia on the first 7 d after AI.