Background: The isolation of non-tuberculous mycobacteria (NTM) has been increasing in South Korea. To date, however, the cause of this increase has not been determined, and it remains unclear whether the use of liquid media has contributed to this increase. The aim of this study was to evaluate the factors associated with NTM isolation and the impact of liquid media on NTM culture. Methods: Mycobacterial smear/culture results of respiratory specimens (sputum and bronchial aspirates), obtained during the years 2002, 2005, and 2010, were retrieved and analyzed retrospectively. Results: During the years 2002, 2005, and 2010, 83,096 sputum specimens were collected from 31,104 patients, and were cultured for mycobacteria, using solid media only in the 2002 and 2005 specimens and both solid and liquid media in the 2010. Of these, 3,516 (4.2%) specimens were smear-positive for acid-fast bacilli (AFB). The annual rate of NTM among positive culture specimens increased from 21% in 2002 to 57.8% in 2010 (p<0.001), as did the proportion of NTM, among AFB smear- and culture-positive specimens, from 12.2% in 2002 to 45.2% in 2010 (p<0.001). In 2010, the NTM culture rate was higher in the liquid than in the solid media (13.9% vs. 8.4%, p<0.001). The NTM rate among AFB-positive specimens was higher in patients aged >50 than ${leq}$50 years. Conclusion: The rate of NTM isolation has steadily been increasing at the hospital in South Korea, likely due in part to the use of liquid media for the culture.