Mitigation of methanogenesis in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal methanogenesis, has a low palatability; therefore, in the present study the aim was to evaluate the mitigation efficacy of its esterified form (monolaurin). Further,¹³C-isotope abundance (δ¹³C) and¹³C–¹²C fractionation during methanogenesis and fermentation were determined to evaluate possible microbial C-isotope preferences. Using the rumen simulation technique, four basal diets, characterised either by the C₃plants grass (hay) and wheat (straw and grain), or the C₄plant (¹³C excess compared with C₃plants) maize (straw and grain), and a mixture of the latter two, were incubated with and without monolaurin (50 g/kg dietary DM). Added to hay, monolaurin did not significantly affect methanogenesis. When added to the other diets (P < 0·05 for the wheat-based diet) methane formation was lowered. Monolaurin decreased fibre disappearance (least effect with the hay diet), acetate:propionate ratio, and protozoal counts. Feed residues and SCFA showed the same δ¹³C as the diets. Methane was depleted in¹³C while CO₂was enriched in¹³C compared with the diets. Monolaurin addition resulted in¹³C depletion of CO₂and enrichment in CH₄(the latter only in the hay diet). In conclusion, monolaurin proved to effectively decrease methanogenesis in the straw–grain diets although this effect might partly be explained by the concomitantly reduced fibre disappearance. The influence on¹³C-isotope abundance and fractionation supports the hypothesis that ruminal microbes seem to differentiate to some extent between C-isotopes during methanogenesis and fermentation.