The present study examined the hypothesis that inhibition of myometrial gap junctions through MAPK1-induced phosphorylation of GJA1 (connexin43) leads to inhibition of spontaneous phasic uterine contractions by 2,2′-dichlorobiphenyl (2,2′-DCB). Uterine strips from Gestation Day 10-pregnant rats exposed in muscle baths to 2,2′-DCB exhibited increased oscillatory frequency and decreased amplitude and synchronization of contractions. To assess effects on gap junctions, Lucifer yellow was injected into myometrial cells and transfer to adjacent cells was scored. After a 1-h treatment, 100 μM 2,2′-DCB decreased Lucifer yellow intercellular transfer in a concentration-dependent manner. The MAP2K1 inhibitor PD98059 increased percentage of dye transfer to adjacent myometrial cells from 18% in cultures exposed for 1 h to 100 μM 2,2′-DCB alone to 48% in cultures cotreated with 50 μM PD98059 and 100 μM 2,2′-DCB. In contrast, the conventional PRKC inhibitor Gö6976 (10 μM) had no significant effect on 2,2′-DCB-induced inhibition of dye transfer. Western blotting showed about a 4.5-fold increase in phosphorylation of GJA1 at S255, a MAPK1 site, after exposure to 100 μM 2,2′-DCB compared to untreated and solvent controls. However, there was no difference in phosphorylation of GJA1 at S368, a PRKC site. Cells treated with 2,2′-DCB increased phosphorylated MAPK1, implicating the increase of activation of MAPK1. Cotreatment with 100 μM 2,2′-DCB and 5 μM PD98059 reversed 2,2′-DCB-induced modification of uterine contractions and increase of pGJA1(S255) in uterine strips. Therefore, this study suggests that 2,2′-DCB decreases amplitude and synchronization of uterine contractions mediated through MAPK1-mediated phosphorylation of GJA1 and subsequent inhibition of myometrial gap junctions.