The objective of this experiment was to study the effects of Brazilian spinach ( Alternanthera sissoo ) pellet (BSP) supplementation on rumen fermentation, protozoal population, and methane (CH 4 ) estimation in beef cattle. Four male Thai native beef cattle, 3 years old, with an average bodyweight of 180 ± 5 kg, were randomly arranged in a 4 × 4 Latin square design. The cattle were supplemented (on-top) with four levels of BSP (2, 4, 6, and 8% dry matter intake (DMI), respectively). The roughage component, derived from rice straw, was fed at 40 % of DMI, while the concentrate diet was fed at 60 % of DMI. The result of the experiment demonstrated that BSP supplementation had no effect on the DMI, nutrient intake, or nutrient digestibility (p > 0.05). Rumen pH and ammonia-nitrogen concentration were not significant, while the average protozoal population linearly decreased (p = 0.002) with BSP supplementation. Mean blood urea-nitrogen concentration was linearly increased (p = 0.004) when increasing the level of BSP. Brazilian spinach pellet had no significant effect on total volatile fatty acids (TVFA), VFA profiles, and CH 4 estimation (p > 0.05). Nitrogen balance was no different from the supplementation of BSP. The study indicates that Brazilian spinach pellet supplementation showed no noticeable effects on feed intake, rumen parameters, and nitrogen utilization; however, at 6-8% of DMI, there was a decrease in protozoal population, with no corresponding reduction in CH 4 estimation.