Our aim was to evaluate the effects of progesterone supplementation after fixed-time artificial insemination (FTAI) on physiological and reproductive variables and serum thyroid hormone concentrations of cooled Holstein heifers during a hot summer season. Sixty-nine Holstein heifers were randomly assigned to three treatments: (1) heifers under visual estrus detection and inseminated according to AM-PM rule (n = 23; control (C)), (2) heifers subjected to FTAI after estrus synchronization using a CIDR insert (n = 24; FTAI group), and (3) heifers treated as the FTAI group plus progesterone supplementation between the day 4 and 14 post-insemination with a reused CIDR device (n = 22; FTAI+SP group). All heifers were cooled daily with misting and fans. Physiological variables were measured; likewise, blood samples were collected to determine serum progesterone, thyroxine, and triiodothyronine concentrations. Both respiration rate and rectal temperature were similar among treatments. Conception rate was greater (P < 0.05) in C (65.2%) and FTAI+SP (59.1%) heifers than in FTAI heifers (33.3%). Serum triiodothyronine and progesterone concentrations of FTAI+SP heifers were similar to those of C heifers but greater (P < 0.05) than those of FTAI heifers. In conclusion, progesterone supplementation post-insemination increased conception rate and serum progesterone concentrations without affecting thermoregulation capacity in Holstein heifers subjected to FTAI and cooling during a hot summer season.