In vitro phosphorylation of 180‐kDa protein, obtained by immunoprecipitation of adipocyte homogenate with anti‐IRS‐1 antibody was increased with the addition of conventional PKC in the presence of Ca^2 +, phosphatidylserine (PS) and diolein (DL). Human purified IRS‐1 was phosphorylated by purified conventional PKC (cPKC) in the presence of Ca^2 +/PS/DL. These results suggest that PKC may have a role in the serine phosphorylation of IRS‐1. In order to clarify the inhibitory effect of cPKC on glucose transport mechanism, we examined the overexpression of PKCβ in cultured adipocytes. Overexpression of PKCβ in adipocytes markedly induced mobility shift and serine phosphorylation of IRS‐1, whereas overexpression of dominant negative PKCβ (DNPKCβ) blocked this mobility shift and serine phosphorylation of IRS‐1. Insulin (10 nM) increased [³H]2‐deoxyglucose (2‐DOG) uptake to 200% from basal level (100%) in cultured adipocytes transfected with a vector alone. Overexpression of PKCβ in adipocytes decreased insulin‐induced 2‐DOG uptake to 110%, whereas overexpression of DNPKCβ increased it to 230%. These results suggest that PKCβ negatively regulates glucose uptake via serine phosphorylation of IRS‐1 in rat adipocytes.