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There is growing evidence that omega-3 (n-3) polyunsaturated fatty-acids (PUFAs) are important for the brain development in childhood and are necessary for an optimal health in adults. However, there have been no studies examining how the n-3 PUFA composition of human milk influences infant behavior or temperament. To fill this knowledge gap, 52 breastfeeding mothers provided milk samples at 3 months postpartum and completed the Infant Behavior Questionnaire (IBQ-R), a widely used parent-report measure of infant temperament. Milk was assessed for n-3 PUFAs and omega-6 (n-6) PUFAs using gas-liquid chromatography. The total fat and the ratio of n-6/n-3 fatty acids in milk were also examined. Linear regression models revealed that infants whose mothers’ milk was richer in n-3 PUFAs had lower scores on the negative affectivity domain of the IBQ-R, a component of temperament associated with a risk for internalizing disorders later in life. These associations remained statistically significant after considering covariates, including maternal age, marital status, and infant birth weight. The n-6 PUFAs, n-6/n-3 ratio, and total fat of milk were not associated with infant temperament. These results suggest that mothers may have the ability to shape the behavior of their offspring by adjusting the n-3 PUFA composition of their milk.