Pair-rule genes are crucial for generating dual segment periodicity for body plan patterning in Drosophila. Bombyx mori is an intermediate germband insect, in which the formation of posterior segments via sequential addition follows a different process from that in Drosophila, although it is somewhat comparable to the process that occurs in vertebrates. Notch signaling is involved in the segmentation of vertebrates, spiders, and basal insects. Groucho (Gro) participates in Notch signaling as a corepressor and plays an important role during segmentation by interacting with other pair-rule proteins. Here, we cloned a gro homolog in the silkworm and positioned it at chromosome 21 in the genetic linkage map. Functional analyses of Bmgro and Bmnotch during embryogenesis were conducted using RNA interference (RNAi). Depletion of Bmgro led to a loss of odd-numbered segments, a characteristic pair-rule phenotype. Bmnotch RNAi resulted in that paired appendages on each segment were symmetrically fused along the ventral midline. An individual segment seemed to possess only one segmental appendage when Notch signaling was compromised. Irregular segments were observed in the Bmnotch RNAi embryo. Our results show that the involvement of Bmgro during the pair-rule process is not mediated by Notch signaling in silkworm. Notch signaling remains in appendage segmentation and restriction of cell fate. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:651–662, 2012. © 2012 Wiley Periodicals, Inc.