Premature delivery remains a serious risk factor in pregnancy, with currently licensed tocolytics unable to offer significant improvement in neonatal outcome. Further understanding of the regulators of uterine contractility is required to enable the development of novel and more effective tocolytic therapies. The transglutaminase family is a class of calcium-dependent, transamidating enzymes, of which tissue transglutaminase 2 is a multifunctional enzyme with roles in cell survival, migration, adhesion, and contractility. The aim of the present study was to investigate the role of this enzyme in regulating the contractility of pregnant human myometrium. Tissue strips from biopsy samples obtained at elective cesarean section were either allowed to contract spontaneously or induced to contract with oxytocin, phenylephrine, or bradykinin. Activity integrals, used to measure contractile activity, were taken following cumulative additions of the reversible, polyamine transglutaminase inhibitors cystamine and mono-dansylcadaverine and the irreversible, site-specific transglutaminase inhibitors N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine and 1,3-dimethyl-2[(oxopropyl)thio]imidazolium. The ability of cystamine and mono-dansylcadaverine to affect oxytocin-mediated calcium mobilization within primary cultured myometrial cells was also measured utilizing a calcium indicator. All inhibitors attenuated myometrial contractions in a concentration-dependent manner independent of the method of contraction stimulus. Similarly cultured myometrial cells preincubated with cystamine and mono-dansylcadaverine displayed an altered calcium response to oxytocin stimulation. Our findings demonstrate a potential role for tissue transglutaminase 2 in regulating uterine contractility in pregnant human myometrium that may be associated with the calcium signaling cascade required for contraction.