The objective of this study was to evaluate the effect of replacing sunflower seed meal (SM) with isonitrogenous Polistes instabilis (PI) on in vitro methanogenesis, rumen fermentation and nutrient digestibility. The SM was replaced with isonitrogenous PI at 0.5, 1, 2, 4 and 8% proportions. Treatment groups were as follows: P0 = no replacement of SM with PI; P0.5 = 0.5% replacement of SM with PI; P1 =1% replacement of SM with PI; P2 = 2% replacement of SM with PI; P4 = 4% replacement of SM with PI; and P8 = 8% replacement of SM with PI. As a result of this study, gas and methane production decreased linearly with PI inclusion in diet. The lowest value of in vitro organic matter digestibility, truly degraded substrate, metabolisable energy, and net energy lactation were observed in the P8 group. In vitro ruminal pH did not change linearly but was higher in P2 group than P0.5 group according to the combined effect. The propionic acid concentration in the rumen was lowest in P1 and P4 group and highest in P8 group; the quadratic effect was also significant. The butyric acid concentration in the rumen was not affected by dietary treatments. The short-chain fatty acids concentration decreased linearly with PI replacement. The fermentation efficiency was higher in P8 group than P1 and P4 groups. The replacement of SM with P. instabilis in beef cattle diet has the potential to reduce methane emissions. However, due to the high lipid content, it generally results in lower gas production and digestibility at 8% inclusion in the diet.