Via bulked segregant analysis sequencing combined with linkage mapping, the ist gene responsible for the irregularly striped rind mutation was delimited to a 144-kb region in cucumber. Sequencing and expression analysis identified Csa1G005490 as the candidate gene. The rind appearance of cucumber is one of the most important commercial quality traits. Usually, an immature cucumber fruit has a uniform rind that varies from green to yellow to white among different cultivated varieties. In the present paper, we isolated a novel fruit appearance cucumber mutant, ist, that has an irregularly striped rind pattern. The mutant displayed green irregular stripes on a yellow-green background at the immature fruit stage. Genetic analysis revealed that a single recessive gene, ist, is responsible for this mutation. A BSA (bulked segregant analysis) sequencing approach combined with genetic mapping delimited the ist locus to an interval with a length of 144 kb, and 21 predicted genes were annotated in the region. Based on mutation site screening and expression analysis, two single-nucleotide polymorphisms within the candidate gene, Csa1G005490, were identified as constituting the mutation. Csa1G005490 encodes a polygalacturonase-1 noncatalytic subunit beta protein (PG1β) known to be involved in fruit softening. The expression of Csa1G005490 was significantly lower in the ist mutant than in the wild type. Transcriptome analysis identified 1796 differentially expressed genes (DEGs) between the ist mutant and wild type. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that these DEGs were enriched mostly in photosynthesis and chlorophyll metabolism pathways. Decreased expression patterns of several chlorophyll synthesis genes in the mutant suggest that ist plays a key role in chlorophyll biosynthesis. These results will provide new insight into the molecular mechanism underlying rind appearance polymorphisms in cucumber.