The aim of this study was to examine the effects of humic substances on fermentation characteristics and microbial communities using the rumen stimulation technique (RUSITEC). The experiment was conducted as a completely randomized design over a 15-d period with 3 treatments duplicated in 2 runs with 2 replicates per run. Treatments consisted of a control diet (forage:concentrate; 60:40) without humic substances or humic substances added at either 1.5 g/d or 3.0 g/d. Dry matter disappearance, pH, fermentation parameters and gas production were measured from d 8 to 15. Samples for microbial profiling were taken on d 5, 10 and 15 using the digested feed bags for solid-associated microbes (SAM) and fermenter fluid for liquid-associated microbes (LAM). The inclusion of humic substances had no effect ( P ≥ 0.19) on DM disappearance, pH or the concentrations of VFA. The production of NH 3 was linearly decreased ( P = 0.04) with increasing levels of humic substances in the diet. There was no effect ( P ≥ 0.43) of humic substances on total gas, CO 2 or CH 4 production. The number of OTUs was significantly reduced in the 3.0 g/d treatment compared to the control on d 10 and 15; however, the microbial community structure was largely unaffected ( P > 0.05). In the SAM samples, the genera Lachnospiraceae XPB1014 group, Succiniclasticum, and Fibrobacter were reduced in the 3.0 g/d treatment and Anaeroplasma, Olsenella, and Pseudobutyrivibrio were increased at d 5, 10, or 15. Within the LAM samples, Christensenellaceae R-7 and Succiniclasticum were the most differentially abundant genera between the control and 3.0 g/d HS treatment samples ( P < 0.05). This study highlights the potential use of humic substances as a natural feed additive which may play a role in nitrogen metabolism without negatively affecting the ruminal microbiota.