Platelet-derived growth factor BB (PDGF-BB) is a Food and Drug Administration (FDA)-approved growth factor, acting as a mitogen and motogen of dermal fibroblasts (DFs), for skin wound healing. The two closely related SH2/SH3 adapter proteins, Nckalpha and Nckbeta, connect PDGF-BB signaling to the actin cytoskeleton and cell motility. The mechanism has not been fully understood. In this study, we investigated, side by side, the roles of Nckalpha and Nckbeta in PDGF-BB-stimulated DF migration. We found that cells expressing the PDGFRbeta-Y751F mutant (preventing Nckalpha binding) or PDGFRbeta-Y1009F mutant (preventing Nckbeta binding), DF cells isolated from Nckalpha- or Nckbeta-knockout mice, and primary human DF cells with RNA interference (RNAi) knockdown of the endogenous Nckalpha or Nckbeta all failed to migrate in response to PDGF-BB. Overexpression of the middle SH3 domain of Nckalpha or Nckbeta alone in human DFs also blocked PDGF-BB-induced cell migration. However, neither Nckalpha nor Nckbeta was required for the activation of the PDGF receptor, p21-activated protein kinase (Pak1), AKT, extracellular signal-regulated kinase (ERK) 1/2, or p38MAP by PDGF-BB. Although PDGF-BB stimulated the membrane translocation of both Nckalpha and Nckbeta, Nckalpha appeared to mediate Cdc42 signaling for filopodium formation, whereas Nckbeta mediated Rho signaling to induce stress fibers. Thus, this study has elucidated the independent roles and mechanisms of action of Nckalpha and Nckbeta in DF migration, which is critical for wound healing.