Induction of the innate immune response in newly hatched chickens is important for limiting infections with bacteria, such as Salmonella enterica serovar Enteriditis (SE). CpG oligodeoxynucleotides (CpG-ODN) can stimulate the innate immune response of young chickens. Therefore, we examined the effectiveness of CpG-ODN administered in ovo on intestinal colonization by SE and the ability to modulate the function of heterophils in young chickens. Heterophils were isolated from 2-day-old chickens and were stimulated with heat-killed SE (HK-SE) or PMA for oxidative burst and HK-SE or live SE for degranulation assays. CpG-ODN treatment had no effect on heterophil oxidative burst when stimulated with HK-SE or PMA. However, HK-SE and live SE increased degranulation (P<0.01) in heterophils from CpG-ODN-treated birds compared to PBS-treated controls. In a second experiment, chickens were orally infected with SE on day 10 post-hatch and cecal contents were collected 6 days later for assessment of SE intestinal colonization. CpG-ODN treatment reduced SE colonization by greater than 10-fold (P<0.001) compared to PBS-injected control birds. Overall, we show for the first time that CpG-ODN given in ovo stimulates innate immune responsiveness of chicken heterophils and increases resistance of young chickens to SE colonization.