Abstract Background When breastfeeding is not possible, infants are fed formulas (IF) in which lipids are usually of plant origin. However, the use of dairy fat in combination with plant oils enables a lipid profile closer to breast milk in terms of fatty acid (FA) composition, triglyceride structure, polar lipids and cholesterol contents. The objective of this study was to determine the effect of an IF containing a mix of dairy fat and plant oils on Omega-3 FA content in red blood cells (RBC). Methods This study was a monocentric, double-blind, controlled, randomized trial. Healthy term infants were fed formulas containing a mix of dairy fat and plant oils (D), plant oils (P) or plant oils supplemented with ARA and DHA (PDHA). Breastfed infants were enrolled as a reference group (BF). FA in RBC phosphatidylethanolamine was evaluated after 4 months and FA in whole blood were evaluated at enrollment and after 4 months by gas chromatography. Differences between groups were assessed using an analysis of covariance with sex and gestational age as covariates. Results Seventy IF-fed and nineteen BF infants completed the protocol. At 4 months, RBC total Omega-3 FA levels in infants fed formula D were significantly higher than in group P and similar to those in groups PDHA and BF. RBC DHA levels in group D were also higher than in group P but lower than in groups PDHA and BF. RBC n-3 DPA levels in group D were higher than in groups P, PDHA and BF. A decrease in proportions of Omega-3 FA in whole blood was observed in all groups. Conclusions A formula containing a mix of dairy lipids and plant oils increased the endogenous conversion of Omega-3 long-chain FA from precursor, leading to higher total Omega-3, DPA and DHA status in RBC than a plant oil-based formula. Modifying lipid quality in IF by adding dairy lipids should be considered as an interesting method to improve Omega-3 FA status. Trial registration Identifier NCT01611649, retrospectively registered on May 25, 2012.