Sex determination in plants gives rise to unisexual flowers.  A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis.  Androecious cucumber (Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production.  The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding.  Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate (EMS)-mutagenized library of the cucumber inbred line ‘406’.  Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11 (ACS11) conferred androecy.  The mutation caused an amino acid change from serine (Ser) to phenylalanine (Phe) at position 301 (S301F).  In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity.  This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.