Abstract: Thirty Holstein cows (10 cows per treatment) were blocked according to calving date, milk yield and party and randomly assigned to a study to determine the effect of manganese (Mn) sources on lactating cows. Treatments were (1) all Mn (14 ppm) supplied by sulfate (S), (2) Mn sulfate (MnSO4) and methionine hydroxyl manganese (Mn-(HMTBA)2) contributed half of the dietary Mn (SM) or (3) all Mn supplied by (Mn-(HMTBA)2) (M). The average 4% Fat-corrected Milk (FCM) yield and fat yield of cows supplied with M dietary were significantly higher than the cows fed with SM and the S (p<0.05). The average lactose rate of SM was significantly lower than S and M (p<0.05). Numerically, the apparent digestibility of organic matter, crude protein, crude fat and acid detergent fiber of S were the lowest but no significant difference were found between groups (p>0.05). Significant increases was observed for serum High-density lipoprotein cholesterol concentration of M compared with S and SM (p<0.05). The average detectable follicle numbers and ovarian score for cows of M was generally lower than cows of SM and S but no significant difference was observed (p>0.10). The present study suggested that replacing a portion of dietary Mn sulfate with Mn-(HMTBA)2 during the peak-and mid-lactation periods will improve the lipid metabolism and milk fat secretion; however, the follicular development may be hampered.