To investigate the function of adaptor protein complex-1 (AP-1) in the silkworm, we characterized AP-1 in the silkworm by RNAi technique and co-localization methods. As a result, AP-1 was found to exist as cytosolic form and membrane-bound form distinguished by phosphate status, showing molecular mass difference. There was relatively more cytosolic form of AP-1 than its membrane-bound counterpart in the silkworm. However, AP-1 distributed predominantly as cytosolic form in BmN cells. Interruption of AP-1 expression via DsRNA was more efficient in BmN cells than in the insect larval, which led to a tendency to dissociation between subcellular organelles like the Golgi apparatus and the mitochondria. Environmental condition changes like relatively higher temperature and treatment with dimethyl sulfoxide can lead to expression variance of AP-1 both in mRNA and protein level. In BmN cells, both the heavy chain γ and light chain σ could clearly co-localize with AP-1 β, mostly forming pits in cytoplasm. Two isoforms of AP-1 σ corresponded to distinct subcellular distribution pattern, possibly due to C-terminal amino acids difference.