Recently, the development of antibiotic resistance emerged as a significant clinical problem in the eradication of Helicobacter pylori. We investigated the MICs of antibiotics for 135 H. pylori isolates from adults in Seoul, South Korea, over the past 16 years. The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and ciprofloxacin increased from 1987 to 2003. Rates of primary resistance to clarithromycin increased from 2.8% in 1994 to 13.8% in 2003. The A2144G mutation was frequently observed in the 23S rRNA gene in clarithromycin-resistant isolates. The increase in resistance to clarithromycin seems to result in a decrease in eradication efficacy for H. pylori. These results suggest that the MICs of several antibiotics for H. pylori have increased over the past 16 years in Seoul.