Starting in 1983, nest boxes for Barn Owls Tyto alba were erected as part of a biological pest control program to deal with rodents, in Kibbutz Sde Eliyahu, and later in other agricultural fields and plantations in the Beit She'an valley, Israel. More than a decade ago, the nest box scheme was extended to include other agricultural areas in the valley, and grew from 14 boxes on 3 km2 in 1983 to about 300 boxes on 90 km2 throughout the entire Beit She'an valley in 2007. Here we present the results of a study during the 2002 through 2006 breeding seasons, in which 156 to 243 nest boxes were monitored each season. Mean occupation of nest boxes during the study was 53.5% (SE 2.1, n = 248), and a total of 596 breeding attempts were recorded, of which 85.2% successfully fledged at least one young. Yearly occupation of nest boxes varied significantly between the years, ranging from 48.1% to 73.5%. The occupancy rate of first-year nest boxes was lower than that of those available for two or more years. The occupancy rate and the number of nestlings per box (per year) were positively correlated to the distance to the closest nest box and negatively correlated to the number of nest boxes within a 500 m radius. Similar to other studies in the world, the erection of nest boxes for Barn Owls in agricultural fields proved extremely successful in the Beit She'an valley, with 86.7% (n = 248) of nest boxes being occupied at least once during the five-year study period. This high occupancy rate demonstrates not only that natural nesting sites were lacking, but also that nest boxes can be used to increase Barn Owl populations in agricultural areas, both for conservation and for biological pest control.