The bacterium causing Johne's disease (JD), Mycobacterium avium subspecies paratuberculosis (MAP), is distributed worldwide and causes progressive intestinal disease, also known as paratuberculosis, in domestic and exotic ruminants, including cattle. The subclinical form of this disease results in progressive weight loss, reduced milk production, lower slaughter value, and premature culling, with possible impacts on fertility and udder health. Eventually, infection can lead to the clinical form that manifests chronic diarrhea, emaciation, debilitation, and eventual death. JD is a major cause of economic loss in cattle industry. In this study, serological testing was conducted by ELISA to determine the prevalence of MAP in Korean native cattle of Jeju during the duration of 2010 to 2013. A total of 5,150 blood sera were collected in 111 farms and the samples were classified into four different age groups for MAP; group 1 (2 years or less), group 2 (2~4 years), group 3 (5~7 years), and group 4 (over 8 years). Overall seroprevalence of MAP was 5.2% (269/5,150), and annually from 2010 to 2013, they were 11.6% (106/915), 2.1% (10/474), 5.5% (89/1,627), and 3.0% (64/2,134), respectively. Also, the prevalence rates was determined by age groups: 48 of group 1 (17.8%), 154 of group 2 (57.3%), 48 of group 3 (17.8%), and 19 of group 4 (7.1%) were seropositive for MAP. Although seropositive samples were found in all age groups, the highest seroprevalence was found in group 2. This study showed that during the investigation the seroprevalence of MAP tended to decrease.