The current study examined lysophospholipids (LPL) on rumen fermentation and bacterial populations in vitro and in vivo. An in vitro batch culture was conducted and the substrate used for the incubation was a ground dairy diet (55% forage and 45% concentrate). Dietary treatments were CON (control), L-LPL, (0.025% LPL in dietary DM; Lipidol Ultra, EASY BIO, Inc.); M-LPL, (0.05%), and H-LPL (0.075%). The incubation was conducted for 48 h in triplicate and the incubation was repeated. All data were analyzed using the MIXED procedure of SAS (incubation as random). Supplementation of LPL tended to increase (linear P = 0.058) propionate as % of total VFA and decrease (linear P = 0.06) A:P ratio. In addition, LPL increased (linearly P < 0.01) ammonia concentration. An in vivo experiment was conducted with 8 cannulated lactating cows in a repeated 4 × 4 Latin square design. Cows were fed a basal diet (CON) supplemented with MON (16 mg/kg in dietary DM), M-LPL or H-LPL. Rumen contents were collected on 2 consecutive days to determine pH, ammonia, VFA, and bacterial population. Bacterial populations were determined using bacterial tag-encoded FLX amplicon pyrosequencing. All data were analyzed using the MIXED procedure of SAS (group and cow within group as random). A decrease (linear P = 0.03) in acetate and numerical increase (P = 0.13) in propionate as % of total VFA resulted in a tendency for a linear decrease (P = 0.09) in the A:P ratio with increasing LPL. Rumen bacterial population was significantly altered by MON vs. CON. However, minimal effects of LPL on microbial population were observed. Increasing LPL in the diet did not affect (P > 0.14) any bacterial populations at phylum level. However, LPL decreased (linear P = 0.05) the proportion of Rikenella and Paludibacter. The proportion of Fibrobacter tended to be greater (P = 0.09) for LPL vs. CON. In conclusion, the pattern of rumen fermentation altered by LPL was generally agreed between in vitro and in vivo. Despite the changes in rumen fermentation by LPL vs. CON, there were minimal effects on bacterial population in the rumen.