In order to enrich the gene pool and the theoretical research of male sterility in cucumber, a male sterility mutant C0128 was used as material for phenotypic identification, genetic analysis and preliminary mapping of male sterility gene. The results showed that: (1) the male petals and anthers of mutant C0128 were smaller than that of wild type CCMC，and no pollen grains were produced on the anthers. (2) Genetic analysis showed that the male sterility of the mutant was controlled by a recessive nuclear gene MS4. (3) Using Gy14×C0128 F₂ population with 1 231 plants and 12 polymorphic SSR markers, the MS4 gene was initially located on cucumber Chr3, and the genetic distance and physical distance was 3.7 cM and 1.21 Mb, respectively. This study confirmed that C0128 is a new male sterile mutant in cucumber. The preliminary mapping results of MS4 can lay a foundation for further cloning of target gene and studying the mechanism of male sterility in C0128.