The purpose of this study was to evaluate the effect of acupuncture versus placebo acupuncture on anxiety in lactating mothers with preterm infants. A parallel, randomized, patient-assessor blind, placebo-controlled trial was conducted in a tertiary school hospital in Londrina, Brazil, between 2011 and 2012. Mothers (n = 29) with very low birth weight infants born at this institution were randomly assigned to two treatment groups: acupuncture, AG (n = 14), or placebo acupuncture, PG (n = 15). Treatment sessions occurred once a week, using 5 Chinese auricular points unilaterally. The primary outcome measure was STAI-State scores, and secondary outcome measure was salivary cortisol levels. Both measures were collected before and after treatment and submitted to a blind assessor. Before-after treatment mean difference in STAI-State scores was observed in both groups (AG = 8.71 and PG = 8.20), not statistically significant (P = 0.888), although within group analysis was significant for both groups (P < 0.005). Salivary cortisol levels did not change after treatment in both groups (P = 0.480). There was no correlation between STAI and salivary cortisol results. At infant's hospital discharge, 76% subjects were breastfeeding exclusively. There was no difference between real and placebo acupuncture for anxiety in mothers with preterm infants.