From an evolutionary perspective, large-scale human groups face an adaptive challenge: maintaining cohesion beyond the limits of one-to-one grooming. Cultural practices that recruit evolved bonding systems may help solve this problem by harnessing mu-opioid psychobiological mechanisms. Religious rituals are cultural practices that consistently promote social cohesion, and here we report a pre-registered large-scale field study examining psychobiological mechanisms underlying these rituals. Participants were tested in the United Kingdom and Brazil at 24 ritual sites (N = 265), with measures taken pre- and post-ritual. Results showed that levels of social bonding (5.4% increase, rR = 0.30) and positive affect (13.1% increase, rR = 0.27) were higher after ritual participation. Importantly, increased social bonding was independently predicted by increases in pain threshold (a proxy for mu-opioid activation; β = 0.13, R2 = 0.02), positive affect (β = 0.25) and connection to a higher power (β = 0.15), even after controlling for country, gender, age and religiosity (R2 = 0.15). This multi-site field study provides experimental yet naturalistic evidence that religious rituals recruit an evolved mu-opioid-based affiliation system that may help sustain cohesion in large congregations, supporting the brain-opioid theory of social attachment.