The stimulus-preceding negativity (SPN) is an event-related potential (ERP) reflecting anticipation. The anterior insular cortex is assumed to be one of the physiological sources of the SPN. However, the precise neural substrates of the SPN have yet to be confirmed. We therefore performed separate functional magnetic resonance imaging (fMRI) and ERP studies using the same time estimation task, followed by fMRI-constrained ERP source analysis. Dipole locations were determined by the fMRI results, while the time courses of dipole activities were modeled by the ERP data. Analysis revealed that the right anterior insula was significantly activated before delivery of the feedback stimulus, whereas the left anterior insula was not, and that the SPN mainly arose from four groups of brain regions related to, respectively: (1) the salience network, (2) reward expectation, (3) perceptual anticipation, and (4) arousal. The results suggest that the SPN pertains to multiple brain functions with complex interactions.