The effects of nano-ZnO on in vitro ruminal fermentation, methane release, total antioxidant capacity (TAC), and microbial biomass production (MBP) were assessed using an in vitro gas production technique. Treatments included a control diet and diets containing 20, 40, or 60 mg of supplemental Zn per kg dry matter (DM) as ZnO or nano-ZnO. As a result of this study, supplementation of 20 mg of Zn as nano-ZnO, similar to ZnO, decreased methane production and protozoa enumeration but improved (P 0.05). The diets containing 40 and 60 mg of supplementary Zn, as nano-ZnO or ZnO, had no advantage over the diet containing 20 mg of Zn in terms of methane decline and TAC, TDS, and MBP increments. Overall, nano-ZnO had no adverse effect on in vitro ruminal fermentation. The addition of 20 mg of Zn as nano-ZnO per kg diet DM was enough to improve the in vitro ruminal fermentation in terms of methane release, TAC, and MBP. Thus, the higher supplementary Zn levels (40 and 60 mg/kg DM) are not recommended.