Background: Intimate social relationships improve individual health and longevity, an effect which is supposed to be mediated through stress-sensitive endocrine and immune mechanisms in response to positive interaction behavior. On a neuroendocrine level, oxytocin (OT) buffers stress responses, modulates social attachment behavior and has been associated with cytokine expression. Consequently, the aim of the present study was to investigate instructed positive couple interaction, observed behavior, and OT in their effect on immune function. Methods: In a 4-group design, 80 healthy couples (N = 160 individuals) received four standard dermal suction blister wounds and were randomized to instructed positive interaction/control and intranasal OT/placebo. Unstimulated cytokines (IL-1β, IL-6, TNF-α) were assessed from wound liquid at 40 min, 105 min and 24 hrs after wounding. Results: Overall, group assignment did not affect friendly or dominant behavior during the interaction sequence. IL-1β and IL-6 levels, however, were moderated by group assignment with lowest levels in women in the positive interaction and OT condition in IL-1 and highest levels in IL-6. TNF-α responses to wounding were not affected from group assignment, however observed friendliness in women was associated with lower TNF-α levels. Discussion: These findings support the immune-regulating role of friendly behavior in romantic couples. Above this, the data provide the first empirical evidence that an intervention that simultaneously targets neuroendocrine mediators and behavior could affect immune function in a sex specific manner and with potential long-term health relevance.