Here, we demonstrated an antagonistic effect of short neuropeptide F (sNPF) in modulating feeding motivation in the silkworm Bombyx mori; sNPF reduced the feeding-delaying effects caused by administration of an inhibitory peptide, allatotropin (AT). In situ hybridization and MALDI-TOF MS analysis revealed the presence of three subtypes of sNPFs (sNPF-1, -2, and -3) in the midgut enteroendocrine cells. Ca²⁺-imaging analyses revealed that three subtypes of sNPF receptors (sNPFRs) (BNGR-A7, -A10, and -A11) showed different affinities with the three subtypes of sNPFs. In addition, sNPF activated its signaling via ERK phosphorylation in the midgut, while mixture of sNPF and AT reduced the phosphorylation level, agreeing with the results of behavioral assay. Together, our current findings suggest that intestinal sNPF positively modulates the feeding motivation by reducing the inhibitory effects by AT within the midgut.