The oxytocin (OT) system, critically involved in social bonding, may also impinge on spirituality, which is the belief in a meaningful life imbued with a sense of connection to a Higher Power and/or the world. Midlife male participants (N = 83) were randomly assigned to receive intranasal OT or placebo. In exploratory analyses, participants were also genotyped for polymorphisms in two genes critical for OT signaling, the oxytocin receptor gene (OXTR rs53576) and CD38 (rs6449182 and rs3796863). Results showed that intranasal OT increased self-reported spirituality on two separate measures and this effect remained significant a week later. It also boosted participants’ experience of specific positive emotions during meditation, at both explicit and implicit levels. Furthermore, the effect of OT on spirituality was moderated by OT-related genotypes. These results provide the first experimental evidence that spirituality, endorsed by millions worldwide, appears to be supported by OT.