AbstractIn this study, the possibility of replacing vegetable fats with ostrich oil in infant formula (IF) production was investigated. The fatty acid profile, the positional distribution of fatty acids in the triacylglycerols, the cholesterol content, and the physicochemical properties of ostrich oil were determined and compared with breast milk fat and vegetable oils. In the next step, two infant formulas were produced using ostrich oil and vegetable oils and the physicochemical properties, rheological properties, color parameters, and sensory analysis of the resultant powders were compared. The results showed that the predominant fatty acids in ostrich oil are palmitic acid, oleic acid, and linoleic acid which is similar to breast milk fat and vegetable oils. The presence of appropriate cholesterol content in ostrich oil makes it more similar to breast milk fat compared to vegetable fats. Palmitic acid was located at sn‐2 position in 15% triacylglycerol from ostrich fat, which was equal to the amount reported for vegetable fats. The incorporation of ostrich oil in infant formula production showed that there is no statistically significant difference between quality attributes of powder formulated with ostrich oil or vegetable oils. Therefore, ostrich oil can be introduced as a new source of edible oil, and addition of ostrich oil is an effective way to reduce the gap between the composition of breast milk and infant formula.