Serial changes of humor comprehension evoked by a well organized four-frame comic Manga were investigated by fMRI in each step of humor comprehension. The neural substrates underlying the amusing effects in response to funny and mixed order manga were compared. In accordance with the time course of the four frames, fMRI activations changed serially. Beginning with the second frame (development scene), activation of the temporo-parietal junction (TPJ) was observed, followed by activations in the temporal and frontal areas during viewing of the third frame (turn scene). For the fourth frame (punch line), strong increased activations were confirmed in the medial prefrontal cortex (MPFC) and cerebellum. Interestingly, distinguishable activation differences in the cerebellum between funny and non-funny conditions were also found for the fourth frame. These findings suggest that humor comprehension evokes activation that initiates in the TPJ and expands to the MPFC and cerebellum at the convergence level.