This is the first report of the diet composition of Long-eared Owl (Asio otus) in the northern and central Negev desert, Israel. The diet consisted of 71.3% small mammals, 26.5% birds, 2.0% invertebrates, and 0.1% reptiles. There were no significant differences among the seven localities studied or among seasons in percent rodents or invertebrates in the diet. However, the proportion of psammophilious rodents within the diet was larger in settlements where the soil was sand or sandy-loess and smaller where the soil was loess or rocky. Percent birds in the diet did not differ among localities, but differed among seasons. Migratory birds formed a significantly larger part of the total birds consumed during migration than during the non-migratory months.