The current study was objected to determine the effect of increased levels of manganese from manganese glycine (Mn-Gly) on ovarian cysts incidence and reproductive performance of multiparous Holstein dairy cows. Five hundred-seven multiparous Holstein cows (178 cows in second parity and 329 cows in third or greater parity) were blocked based on parity (2 or ≥ 3), body condition score (BCS), and previous lactation milk yield and randomly allocated to one of three treatments providing 0 (CON; n = 180), 40 (40 Mn; n = 178), and 80 (80 Mn; n = 149) mg of supplemental Mn/kg of DM from Mn-Gly. The experimental treatments were fed from calving to 100 DIM, and the CON diet in fresh and high-producing cows contained 64 and 46 mg of Mn/kg DM as MnSO4, respectively. Blood samples were taken from 50 cows in each treatment at 0, 14, 28, 42, 60, and 90 DIM for serum metabolites determination. A subset of 12 cows per treatment group was randomly chosen to measure whole blood concentration of Mn at 0 and 90 DIM. Milk yield and composition were recorded monthly up to 150 DIM. The evaluation of BCS was performed monthly. Ovaries from 50 cows per treatment were examined by transrectal ultrasonography at 43 ± 2 DIM to monitor ovarian structures. Days in milk at first service, pregnancy per artificial insemination (P/AI) at first and second service, pregnancy rate at 150 DIM, the number of service per conception, and days to pregnancy were recorded. Within the first 60 DIM, 24 cows were culled and no treatment effect was noted (P = 0.66). Relative to CON, cows fed 80 Mn had lower ovarian cysts incidence (P = 0.05; 17.5 ± 6.2 vs. 2.0 ± 1.9 %), and 40Mn cows tended to be have decreased ovarian cysts incidence (P = 0.1; 17.5 ± 6.2 vs. 7.6 ± 4.0 %). The percentage of follicles ≥ 3 mm diameter was not affected by treatments (P = 0.18). Cows fed 40Mn and 80Mn treatments tended (P = 0.08) to increase corpus luteum (CL) percentage on ovaries than CON treatment (72.0 ± 7.1 and 76.0 ± 6.7 vs. 56.0 ± 8.1%). The concentrations of serum glucose (P < 0.01) and cholesterol (P < 0.01) was enhanced by Mn supplementation to diets, but serum calcium, magnesium, and phosphorus concentrations were not influenced by treatments (P> 0.1). No treatments effect were observed for days in milk at first service, P/AI at the first service, pregnancy rate at 150 DIM, the number of service per conception, and days open (P > 0.1). However, P/AI at the second service tended (P = 0.07) to increase by Mn supplementation to diets, as second service P/AI was 9.1 and 15.8% greater for 40 Mn and 80 Mn cows than CON cows. The changes of BCS were affected by treatments (P = 0.02), as the 80 Mn cows had smaller body condition losses during experimental period. Yield of milk tended (P = 0.1) to be higher for 40 Mn compared with CON (47.67, 49.0, and 48.71 kg/day for CON, 40 Mn and 80 Mn, respectively), but no difference was found between 80 Mn and CON. Increasing the levels of Mn in the diets (P < 0.01) reduced milk fat and protein content. In general, supplementing the diet with 80 mg Mn/kg DM using Mn-Gly decreased the incidence of ovarian cysts, increased the presence of CL on the ovaries and improved second- service P/AI.