A 14 bp deletion in CsACS2 gene encoding a truncated loss-of-function protein is responsible for elongated fruit shape and perfect flowers in cucumber. In cucumber (Cucumis sativus L.), sex expression and fruit shape are important components of biological and marketable yield. The association of fruit shape and sex expression is a very interesting phenomenon. The sex determination is controlled primarily by the F (female) and M (monoecy) loci. Homozygous recessive mm plants bear bisexual (perfect) flowers, and the fruits are often round shaped. CsACS2 encoding the 1-aminocyclopropane-1-carboxylic acid synthase has been shown to be the candidate gene for the m locus. We recently identified an andromonoecious cucumber line H38 that has bisexual flowers but elongated fruits. To rapidly clone this monoecious gene in H38, we developed a tri-parent mapping strategy, which took advantage of the high-density Gy14 × 9930 cucumber genetic map and the powder of bulk segregant analysis. Microsatellite markers from the Gy14 × 9930 map were used to screen two pairs of unisexual and bisexual bulks constructed from H38 × Gy14 and H38 × 9930 F2 populations. Polymorphic markers were identified and used to quickly develop a framework map and place the monoecious locus of H38 in cucumber chromosome 1. Further fine mapping allowed identification of a novel allele, m-1, at the monoecious locus to control the bisexual flower in H38, which was due to a 14 bp deletion in the third exon of the CsACS2 gene encoding a truncated loss-of-function protein of the cucumber 1-aminocyclopropane-1-carboxylic acid synthase. This new allele provides a valuable tool in understanding the molecular mechanisms of CsACS2 in the relationships of sex determination, fruit shape, and CsACS activities in cucumber.