Objective Levosimendan, a compound that exerts effects on calcium sensitivity and intracellular free calcium, in addition to opening ATP-sensitive K-channels, is widely used in the treatment of heart failure. Because of its dual mechanism of action, we hypothesized that it would modulate human uterine contractility. Study Design Biopsies of human myometrium were obtained at elective cesarean section (n = 16). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of 1 nmol/L to 100 mmol/L. In separate experiments, the effects of prior exposure to the K-ATP antagonist glibenclmide (100 mmol) on the effects of levosimendan on myometrial contractility were evaluated. Simultaneous controls were performed for all experiments. Results Levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, when compared with control strips. The mean maximal inhibition (MMI) values were as follows: 45.34% ± 5.92% for spontaneous contractions (n = 6; P < .05), and 41.88% ± 5.40% for oxytocin-induced contractions (n = 6; P < .05). The inhibitory effect of levosimendan was significantly antagonized by glibenclamide, resulting in the mean maximal inhibition for levosimendan reduced to 19.04% ± 3.61% for spontaneous contractions (n = 6; P < .05), and 16.53% ± 4.08% for oxytocin induced contractions (n = 6; P < .05). Conclusion Levosimendan exerted a potent relaxant effect on spontaneous and agonist-induced human uterine contractility in vitro. This effect was reduced in the presence of K-ATP blockade. Because of the putative role of levosimendan in inflammatory conditions, the findings here may have implications for its future use as therapy for preterm labor.