We investigated the effects of maternal docosahexanoic acid (DHA) supplementation on pups’ auditory startle responses and the composition of brain myelin. Methods: Timed-pregnant rats were fed throughout pregnancy and lactation diets that contained 0, 0.3, 0.7 or 3% of total fatty acids as DHA. Milk was collected from culled pups’ stomachs on postnatal day (PND) 3, latency of the auditory startle reflex was measured on PND 15, and pups were killed and brains collected on PND 24. Results: Higher levels of DHA in maternal diet were reflected in milk and in pups’ myelin. The latency of the auditory startle response was significantly longer in offspring of dams fed higher levels of DHA. There was a positive correlation between the myelin content of DHA and the latency of the startle response (p = 0.044), and a negative correlation between the myelin content of DHA and the myelin content of cholesterol (p = 0.005). Conclusion: High levels of maternal DHA intake alter the lipid composition of rat pup myelin, and are associated with longer latencies of the auditory startle response – a myelin-dependent electrophysiologic response.