Enhancement of alcohol tolerance in microorganisms is an important strategy for improving bioalcohol productivity. Although cyanobacteria can be used as a promising biocatalyst to produce various alcohols directly from CO2, low productivity, and low tolerance against alcohols are the main issues to be resolved. Nevertheless, to date, a mutant with increasing alcohol tolerance has rarely been reported. In this study, we attempted to select isopropanol (IPA)-tolerant mutants of Synechococcus elongatus PCC 7942 using UV-C-induced random mutagenesis, followed by enrichment of the tolerant candidates in medium containing 10g/L IPA and screening of the cells with a high growth rate in the single cell culture system in liquid medium containing 10g/L IPA. We successfully acquired the most tolerant strain, SY1043, which maintains the ability to grow in medium containing 30g/L IPA. The photosynthetic oxygen-evolving activities of SY1043 were almost same in cells after 72h incubation under light with or without 10g/L IPA, while the activity of the wild-type was remarkably decreased after the incubation with IPA. SY1043 also showed higher tolerance to ethanol, 1-butanol, isobutanol, and 1-pentanol than the wild type. These results suggest that SY1043 would be a promising candidate to improve alcohol production using cyanobacteria. Biotechnol. Bioeng. 2017;114: 1771-1778. (c) 2017 Wiley Periodicals, Inc.