The transition of the sexual mode occurs widely in animal evolution. In Caenorhabditis nematodes, androdioecy, a sexual polymorphism composed of males and hermaphrodites having the ability to self-fertilize, has evolved independently multiple times. Although regulatory elements in non-coding regions are likely involved in the evolution of hermaphroditism, their contribution to the transition of sexual mode is ambiguous. Here, we conducted a genome-wide analysis of conserved non-coding elements (CNEs) focusing on the evolution of hermaphroditism in Caenorhabditis nematodes. We found that, in androdioecious nematodes, mutations rapidly accumulated in CNEs neighboring genes associated with sexual traits. Expression analysis indicate that the identified CNEs are involved in spermatogenesis in hermaphrodites and associated with the transition of gene expression from dioecious to androdioecious nematodes. Finally, genome editing of a CNE neighboring laf-1 resulted in a change in its expression in the gonadal region undergoing spermatogenesis. Our bioinformatic and experimental analyses highlight the importance of CNEs to the transition of sexual mode.