In nature, most self-incompatible flowering plants (angiosperms) show gametophytic self-incompatibility. Although gametophytic self-incompatibility functions can ultimately prevent self-fertilization, flowering plants have adopted different signal transduction pathways to reject self pollen. At present, there are mainly two pathways of signal transduction on gametophytic self-incompatibility. One is the S-RNase-based signal transduction in Solanaceae, Scrophulariaceae, and Rosaceae. The other is the cytosolic free Ca2+ acting as a second messenger in pollen of Papaveraceae. This review highlights the recent progress made towards understanding the signal transduction on gametophytic self-incompatibility.