Abstract Background Methylnicotinamide (MNA) displays vasoprotective activity, however, the regulation of the activity of nicotin-amide-N-methyltransferase (NNMT), is largely unknown. We analyze a possible involvement of IL-6 in the activation of NNMTMNA pathway during an endurance exercise. Methods FVB, C57Bl/6J IL6+/+ and C57Bl/6J IL-6−/− mice were subjected to the single bout of endurance exercise consisting of 90min of swimming. Thereafter, exercise-induced changes in NNMT activity in the liver as well as concomitant changes in the concentration of MNA and its further metabolites in plasma were analyzed. Results In two strains of mice (FVB and C57Bl/6J IL6+/+) 90min of swimming resulted in approximately 2–3 folds increase in NNMT activity (from 0.14± 0.03to 0.421± 0.02pmol/min/mg, p<0.05 and from 0.2± 0.06to 0.35± 0.07pmol/min/mg, p<0.01, respectively) and concomitant increase in the plasma concentration of MNA (from 157± 15.06 to 230± 16.2ng/ml, p<0.01, and from 77.05± 14.06ng/ml to 152.55± 58.4ng/ml; p<0.01, respectively). However, in C57Bl/6J IL-6−/− mice 90min of swimming did not change liver NNMT activity (from 0.25 ± 0.07 to 0.23 ± 0.06pmol/min/mg), while MNA concentration in plasma rose approximately two-fold (from 65.3± 30.9ng/ml to 124.8± 35.8ng/ml; p<0.05). Conclusions We demonstrated for the first time that NNMT – MNA pathway is activated by a single bout of endurance exercise. Interestingly, exercise-induced activation of NNMT in the liver involves IL-6, while the rise in MNA concentration in plasma was partially IL-6-independent. Taking into the consideration the pharmacological activity of MNA, IL-6-dependent and IL-6-independent activation of NNMT, may contribute to the exercise capacity. The physiological role of NNMT in the exercise warrant further studies.