Abstract The emergence of unisexual flower is an important event during plant evolution. The molecular mechanism underlying the formation of unisexual flowers remains unclear in dioecious spinach. In this study, we identified the spinach MALE STERILITY1 gene, SpMS1, which serves as a masculine factor to regulate male fertility and sex reversion. Silencing SpMS1 led to stamen sterility in male flowers and the development of masculine traits in female flowers. Overexpression of SpMS1 in wild-type Arabidopsis resulted in sterile stamens and irregular pollen exine. Notably, ectopic expression of SpMS1 in Arabidopsis ms1 mutants restored pollen viability and flower fertility. Furthermore, our findings demonstrate that SpMS1 interacts with MADS-box transcription factor SpAP1 to regulate unisexual flower development. Thus, SpMS1 exhibits a conserved function in pollen fertility akin to bisexual flowers, while also acting as a key regulator of unisexual flower development in spinach. This study sheds light on the mechanism of sex differentiation in dioecious plants and also provides valuable insights for manipulating male sterility in plant breeding programs.