We evaluated whether ELISPOT assay can predict tuberculosis (TB) development in kidney‐transplantation (KT) recipients with a negative tuberculin skin test (TST). All adult patients admitted to a KT institute between June 2008 and December 2009 were enrolled; TB development after KT was observed between June 2008 and December 2010. Isoniazid (INH) was given to those patients with positive TST or clinical risk factors for latent TB infection (LTBI). ELISPOT assay was performed on all patients, and TB development after KT was observed by a researcher blinded to the results of ELISPOT. A total of 312 KT recipients including 242 (78%) living‐donor KT were enrolled. Of the 312 patients, 40 (13%) had positive TST or clinical risk factors for LTBI and received INH; none developed TB after KT. Of the remaining 272 patients, 4 (6%) of 71 with positive ELISPOT assay developed TB after KT, whereas none of the 201 patients with negative (n = 171) or indeterminate ELISPOTs (n = 30) developed TB after KT (rate difference between positive and negative/indeterminate ELISPOT, 3.3 per 100 person‐years 95% CI 1.4–5.1, p<0.001). Positive ELISPOT results predict subsequent development of TB in KT recipients in whom LTBI cannot be detected by TST or who lack clinical risk factors for LTBI. Positive results of an interferon‐gamma releasing assay anticipate the subsequent development of tuberculosis in kidney transplant recipients in whom latent tuberculosis infection cannot be detected by tuberculin skin test or who lack clinical risk factors for latent tuberculosis infection. See editorial by Torre‐Cisneros and Doblas on page 1769.