Pneumonia caused by Rhodococcus equi, a facultative, intracellular bacterium that replicates within macrophages, is an important cause of disease and death in immunocompromised hosts. No vaccine is yet available, and control is based on treatment with antibiotics. The emergence of antibiotic resistance and the lack of effective alternative antimicrobials indicate novel approaches to control are needed, such as host-directed strategies. Cytosine-phosphate-guanine oligodeoxynucleotides (CpGs), Toll-like receptor (TLR) 9 agonists, have immunomodulatory function both in vitro and in vivo in foals. Our objective was to evaluate the impact of in vitro treatment of neonatal foal alveolar macrophages (AMs) with CpG 2142 during the first month after birth. Using broncho-alveolar lavage, AMs were collected from 6 foals at ages 2, 14, and 26 days. Effects of age and treatment on the intracellular killing of R. equi by foal AMs were determined using the ratio of colony-forming units at 48 hours (T48) to post-phagocytosis (T0) (i.e, T48:T0). Foal AMs had significantly (P > 0.05) greater killing capacity at 14 and 26 days than at 2 days of age. Relative to media control, CpG 2142 increased the killing capacity of R. equi by foal AMs at ages 2, 14, and 26 days by approximately 2, 10, and 8 fold (P > 0.05), respectively. Notably, CpG 2142 clearly enhanced killing of R. equi by foal AMs even at age 2 days, when neonatal foals are known to lack a fully functional immune system. CpG 2142 can stimulate innate immune cells including AMs to kill intracellular pathogens more efficiently, and might be effective when nebulized for host-directed therapy against pneumonia caused by R. equi and similar intracellular pathogens such as Mycobacterium tuberculosis.