The determinate growth habit of plants reduces the number of internodes and shortens the main stem by terminating the shoot apical meristem through a transition to inflorescence. Understanding the genetic basis of this habit can help optimize crop yield and cultivation technology for vegetable breeding. This study aimed to identify the determinate-2 (de-2) gene responsible for the determinate growth habit in the W-sk cucumber line. Termination of the main stem in the W-sk line occurred between 14 and 23 internodes, depending on cultivation conditions. Resequencing of the W-sk genome identified a novel SNP in the cucumber TERMINAL FLOWER1 (CsTFL1) gene, explaining the de-2 phenotype. This was verified with a CAPS-T marker cosegregation with determinate growth in the F2:3 population, and this polymorphism is unique among genotyped indeterminate cucumber cultivars or breeding lines. Crossing the W-sk line with the G421 line with the determinate (de) gene confirmed the allelism of both genes. An SNP in CsTFL1 in the W-sk line introduced a premature stop codon, resulting in the putative deletion of 13 amino acids, possibly causing determinate growth habit. Overall, this study provides insights into the genetic basis of cucumber plant growth architecture and advances in cucumber breeding.