The aim was to elucidate the impact of oral folic acid (FA) supplements on progesterone profile, blood metabolites and biochemical indices of heat-stressed Holstein cows during the early stage of pregnancy. The study lasted from the day of artificial insemination through the end of the fourth week of pregnancy. The first group (CON, n=17) received 0 µg of FA/Kg BW as a control. The second and third groups received oral FA doses of 5 (FA5, n=19) and 10 (FA10, n=20) µg/Kg BW, respectively. At the 2nd and 3rd weeks of pregnancy, the FA10 group had greater progesterone levels than the CON group (P<0.05). The FA10 group had a greater progesterone level than the FA5 and CON groups at the fourth week of pregnancy (P<0.01). The FA10 group had higher folate levels than CON group during the first three weeks of pregnancy (P<0.01). Both FA-supplemented groups had significantly greater serum folates than the CON group by the end of the fourth week of pregnancy (P<0.01). At the 2nd and 4th weeks of pregnancy, the FA10 group had greater levels of serum glucose and globulin than the CON group (P=0.028 and 0.049, respectively). Both FA-supplemented groups had greater serum growth hormone (GH) levels at the 4th week of pregnancy (P=0.020). Additionally, the FA10 group showed significantly higher levels of IGF-1 at the 2nd and 4th week of gestation (P=0.040 and 0.001, respectively). FA supplementation decreased the levels of non-esterified fatty acid (NEFA) at the 2nd and 4th week of gestation (P=0.020 and 0.035, respectively). Additionally, the FA10 group showed significantly higher pregnancy-associated glycoprotein (PAG) levels at the 2nd and 4th week of gestation (P=0.005 and 0.001, respectively). In conclusion, oral FA supplementation (10 µg/Kg BW) in the first month of gestation improved the progesterone profile, as well as blood folates, PAG, GH, and IGF-1 concentrations in heat-stressed Holstein cows. These findings could be useful in developing practical strategies to keep dairy cows’ regular reproductive patterns under heat stress conditions.