An experiment was conducted on 3 male rumen fistulated adult buffaloes fed on wheaten straw and concentrate mixture in a Latin square design to study the impact of niacin supplementation on rumen metabolites. Three animals were fed wheaten straw+concentrate mixture (group I, control), wheaten straw+concentrate mixture+100 ppm niacin (group II), and wheaten straw +concentrate mixture+200 ppm niacin (group III). After 21 days feeding, rumen liquor was drawn for 3 consecutive days at different time intervals (0, 2, 4, 6 and 8 h) to study the various rumen metabolites i.e., rumen pH ammonia-N, total-N, trichloroacetic acid precipitable-N, non-protein nitrogen, total volatile fatty acids, their fractions and number of protozoa. Mean pH values in strained rumen liquor (SRL) of animals in 3 groups were 6.64, 6.71 and 6.67, indicating no statistically significant difference. Results revealed a significant (p < 0.01) increase in TVFA concentration among the supplemented groups (group II and III) in comparison to control group. Mean TVFA concentration (meq/dl) was 9.75, 10.97 and 11.44 in 3 groups respectively The highest concentration of TVFA was observed at 4 h and minimum at 0 h in all the 3 groups. The percentage of acetic, propionic, butyric and isobutyric acid was statistically similar among the three groups. The mean ammonia-N concentration (mg/dl SRL) was significantly (p < 0.01) lower in group II (16.38) and group III (15.42) than group I (18.14). Ammonia-N concentration was higher (p < 0.01) at 4 h as compared to all the time intervals. The mean total-N concentration (mg/dl SRL) was higher (p < 0.01) in group II (74.16) and group III (75.47) as compared to group I (62.04). Total-N concentration was higher (p < 0.01) at 4 h as compared to other time intervals and lowest value was recorded at 0 h.Concentration of TCA-ppt-N (mg/dl SRL) was significantly (p < 0.01) lower in control group as compared to niacin supplemented groups. Mean value of NPN (mg/dl SRL) was significantly (p < 0.01) lower in group III (23.21) as compared to group I (25.71), whereas groups I and II, and groups II and III were similar to each other. Total protozoa number (x 10(4)/rr.l SRL) ranged from 18.06 to 27.41 in group 1, 20.89 to 38.44 in group II and 27.61 to 39.45 in group III. The mean protozoa number was significantly (p < 0.01) higher in SRL of group II (27.60) and III (30.59) as compared to group I (22.48). It can be concluded from the study that supplementation of niacin in the diet of buffaloes had improved the rumen fermentation by decreasing the concentration of ammonia-N and increasing protein synthesis.