BSA-seq combined with whole-genome resequencing map-based cloning delimited the cucumber det-novel locus into a 44.5 kb region in chromosome 6 harboring a putative candidate gene encoding a phosphatidylethanolamine-binding protein (CsCEN). Determinate and indeterminate growth habits of cucumber can affect plant architecture and crop yield. The TERMINAL FLOWER 1 (TFL1) controls determinate/indeterminate growth in Arabidopsis. In this study, a novel mutation in cucumber TFL1 homolog (CsCEN) has shown to regulate determinate growth and product of terminal flowers in cucumber (Cucumis sativus L.), which is similar to the function of CsTFL1 as previously reported. Genetic analysis in two determinate genotypes (D226 and D082) and indeterminate genotype (CCMC) revealed that a single recessive gene is responsible for this determinate growth trait. With the combination of BSA-seq and whole-genome resequencing, the locus of determinate-novel (det-novel) trait was mapped to a 44.5 kb genomic region in chromosome 6. Sequence alignment identified one non-synonymous SNP mutation (A to T) in the third exon of CsCEN, resulting in an amino acid substitution (Thr to Pro), suggesting that determinate growth might be controlled by a novel gene CsCEN (Csa6G152360) which differed from the reported CsTFL1 gene. The CsCEN expression level in shoot apexes and axillary buds was significantly lower in D226 compared to CCMC, suggesting its essential role in sustaining indeterminate growth habit. Identification and characterization of the CsCEN in the present study provide a new insight into plant architecture modification and development of cucumber cultivars suited to mechanized production system.