RhoA is a key player in actin cytoskeleton reorganization and exerts most of its effect through the RhoA-ROCKs signaling pathway. Although recent studies have stressed the roles of ROCKs as regulators of glucose metabolism, little is known of the roles played by RhoA, the upstream regulators of ROCKs and other isotypes of Rho small-GTPases. This study was undertaken to determine whether Rho isotypes modulate glucose transport and insulin signaling in insulin-sensitive cell models, that is, 3T3-L1 adipocytes and L6 myoblasts. Glucose uptake assays showed that RhoA knockdown using siRNA reduced insulin-stimulated glucose transport in both cell types, whereas knockdown of RhoB or RhoC did not. Furthermore, RhoA overexpression increased insulin-stimulated glucose transport. Interestingly, the insulin-stimulated PI3K-Akt signaling pathway was unaffected under RhoA-depleted or -overexpressed conditions, which suggested RhoA might regulate glucose transport via an Akt-independent pathway. Interestingly, an immunoblot assay of signaling molecules related to actin-myosin cytoskeletal remodeling showed that unlike RhoA or RhoC, RhoA regulated ERM phosphorylation. Our results suggest that RhoA, but not RhoB or RhoC, mediates glucose transport by regulating the vesicle trafficking machinery in an Akt-independent manner. (C) 2019 Elsevier Inc. All rights reserved.