The microspore can follow two different developmental pathways. In vivo microspores follow the gametophytic program to produce pollen grains. In vitro, isolated microspores can be reprogrammed by stress treatments and follow the embryogenic program, producing doubled-haploid embryos. In the present study, we analyzed the dynamics and role of endogenous auxin in microspore development during these two different scenarios, inBrassica napus.We analyzed auxin concentration, cellular accumulation, the expression of theTAA1auxin biosynthesis gene, and thePIN1-likeefflux carrier gene, as well as the effects of inhibiting auxin biosynthesis by kynurenine on microspore embryogenesis. During the gametophytic pathway, auxin levels andTAA1andPIN1-likeexpression were high at early stages, in tetrads and tapetum, while they progressively decreased during gametogenesis in both pollen and tapetum cells. In contrast, in microspore embryogenesis,TAA1andPIN1-likegenes were upregulated, and auxin concentration increased from the first embryogenic divisions. Kynurenine treatment decreased both embryogenesis induction and embryo production, indicating that auxin biosynthesis is required for microspore embryogenesis initiation and progression. The findings indicate that auxin exhibits two opposite profiles during these two microspore developmental pathways, which determine the different cell fates of the microspore.