Mycobacterium avium subsp. paratuberculosis (MAP) was isolated from a Hog deer, died of accident in Mathura district. Necropsy was conducted at Veterinary College Mathura and the tissue samples (Mesenteric lymph nodes and intestine near ileo-cecal junction), and fecal samples were collected for isolation of MAP by direct microscopy, culture. Hog deer was extremely healthy and tissues (MLN and intestine) did not show any gross pathological lesions suggestive of Johne's disease and were normal and not swollen or enlarged. Tissues and fecal samples on screening by culture on HEY medium with mycobactin J showed that only the small intestine tissues collected from ileo caecal junction were positive for MAP bacilli. Direct microscopy of the intestinal tissues revealed abundant acid-fast bacilli similar to MAP. Colonies appeared in all tubes around 36 days of incubation. Colonies were characterized on the basis of slow growth, mycobactin J dependency and acid fastness.