Abstract This study aimed to evaluate the effect of nano-encapsulation of four essential amino acids (AA), threonine, methionine, tryptophan, and lysine on in vitro ruminal total gas, methane, carbon monoxide, and hydrogen sulfide production as well as the rumen fermentation profile in cattle. The highest (P lysine > tryptophan > methionine (P < 0.0001) and the rate of production per hour followed the same trend (P = 0.0259). CH4 parameters showed that in 4 h, 24 h, and 48 h of incubation the lowest methane production was obtained in the diet with methionine (P < 0.05) and the highest one in diet supplemented with threonine. Methane fractions showed that methionine-containing diets resulted in more (P < 0.05) metabolizable energy versus methane, followed by tryptophan-containing, and then lysine-containing diets. Methionine-fortified diets seem to be the most eco-friendly among those studied regarding methane output. However, based on methane, CO, and H2S output as well as the rumen fermentation profile nano-encapsulated lysine is recommended for use in ruminant nutrition.