In recent years, Crohn's disease (CD, chronic, relapsing inflammatory condition of bowel) has been reported with increasing frequency in India. However, etiology of CD still remains obscure. Present study investigated association of Mycobacterium avium subsp. paratuberculosis (MAP) in cases of CD using microscopic examination, culture, ELISA and PCR tests. A total of 18 samples (5 stool, 5 biopsies, 5 serum and 3 blood clot) were collected from 5 patients, diagnosed suffering with CD in the Gastroenterology department of All India Institute for Medical Sciences (AIIMS), New Delhi. Samples were screened for the presence of MAP and its antibodies. Isolation of MAP was done as per method of Whipple et al., (1991) with some modifications (Singh et al., 1998), on HEY medium with Mycobactin J. ELISA was performed as per method described by Singh et al., (2007) with some modification. Antigens derived from MAP 'Bison type' (S 5) of goat origin and MAP 'Bovine' strain were used at 0.1 μg / well and 2 μg / well concentration in ELISA, respectively. None of the stool, biopsies and blood clot sample were positive for acid-fast bacilli. Of the 5 CD patients, 4 (80.0%) were positive for MAP in culture of both biopsies and stool samples. MAP was isolated from 2 (66.6%) of 3 blood clots left after harvesting serum from CD patients. Same individual were also positive for MAP in stool and biopsies cultures. Majority of MAP colonies grew around 120 days of incubation on Herrold's egg yolk medium (HEYM) with mycobactin J (Allied Monitor, Inc, USA). Primary colonies were identified on the basis of cultural characteristics, bacterial morphology, slow growth, mycobactin J dependency, acid fastness and finally IS900 PCR. All CD patients were positive for MAP antibodies using antigen from MAP 'Bison type' strain. Using MAP 'Bovine' antigen detected only 2 (40%) of 5 CD patients positive for MAP. 'Bison type' antigen had better correlation with culture (stool and biopsies) from CD patients. Present study indicated that MAP may play an important role in the pathogenesis of CD, ELISA developed using MAP 'Bison type' S 5 antigen can be used as mass screening test for MAP infection in human. The study is the first report of association of patients MAP with CD in human beings, in India.