The naturally occurring bisexual cone of gymnosperms is considered to have been a potential intermediate stage in the origin of flowers, but the mechanisms governing bisexual cone formation remain largely elusive. Here, we employed transcriptomic and DNA methylomic analyses, together with hormone measurement, to investigate the molecular mechanisms underlying bisexual cone development in a conifer species Picea crassifolia. Our study reveals a “bisexual” expression profile in bisexual cones, especially in expression patterns of B-, C-class and LEAFY genes, supporting the out of male model. GGM7 could be essential for initiating bisexual cones. DNA methylation reconfiguration in bisexual cones affects the expression of genes crucial for cone development, including PcDAL12, PcDAL10, PcNEEDLY and PcHDG5. Auxin likely plays an important role in the development of female structures of bisexual cones. This study unveils the potential mechanisms responsible for bisexual cone formation in conifers and may shed light on the development of bisexuality.