Multiple pregnancy is a major cause of spontaneous preterm birth, which is related to uterine overdistention. The objective of this study was to determine whether an oxytocin receptor antagonist, retosiban (GSK221149A), inhibited the procontractile effect of stretch on human myometrium. Myometrial biopsies were obtained at term planned cesarean delivery (n = 12). Each biopsy specimen was dissected into 8 strips that were exposed in pairs to low or high stretch (0.6 or 2.4 g) in the presence of retosiban (1 μM) or vehicle (dimethylsulfoxide) for 24 hours. Subsequently, we analyzed the contractile responses to KCl and oxytocin in the absence of retosiban. We found that incubation under high stretch in vehicle alone increased the response of myometrial explants to both KCl (P = .007) and oxytocin (P = .01). However, there was no statistically significant effect of stretch when explants were incubated with retosiban (P = .3 and .2, respectively). Incubation with retosiban in low stretch had no statistically significant effect on the response to either KCl or oxytocin (P = .8 and >.9, respectively). Incubation with retosiban in high stretch resulted in a statistically significant reduction (median fold change, interquartile range, P) in the response to both KCl (0.74, 0.60-1.03, P = .046) and oxytocin (0.71, 0.53-0.91, P = .008). The greater the effect of stretch on explants from a given patient, the greater was the inhibitory effect of retosiban (r = -0.65, P = .02 for KCl and r= -0.73, P = .007 for oxytocin). These results suggest that retosiban prevented stretch-induced stimulation of human myometrial contractility. Retosiban treatment is a potential approach for preventing preterm birth in multiple pregnancy.