Oxytocin-induced Ca2+ transients play an important role in myometrial contractions. Here, using a knockout model, we found that the enzyme CD38, responsible for the synthesis of the second messenger cyclic ADP-ribose (cADPR), plays an important role in the oxytocin-induced Ca2+ transients and contraction. We also observed that CD38 is necessary for TNF-α-increased agonist-stimulated Ca2+ transients in human myometrial cells. We provide experimental evidence that the TNF-α effect is mediated by increased expression of the enzyme CD38. First, we observed that TNF-α increased oxytocin-induced Ca2+ transients and CD38 expression in human myometrial cells. Moreover, using small interference RNA technology, we observed that TNF-α stimulation of agonist-induced Ca 2+ transients was abolished by blocking the expression of CD38. In control experiments, we observed that activation of the component of the TNF-α signaling pathway, NF-κB, was not affected by the treatments. Finally, we observed that the effects of TNF-α on CD38 cyclase and oxytocin-induced Ca2+ transients are abolished by progesterone. In conclusion, we provide the first experimental evidence that CD38 is important for myometrial Ca2+ transients and contraction. Moreover, CD38 is necessary for the TNF-α-mediated augmentation of agonist-induced Ca 2+ transients in myometrial cells. We propose that the balance between cytokines and placental steroids regulates the expression of CD38 in vivo and cell responsiveness to oxytocin.