Human keratinocytes (HK) migration plays a critical role in the re-epithelialization of acute skin wounds. Although extracellular matrices (ECM) and growth factors (GF) are the two major pro-motility signals, their functional relationship remains unclear. We investigated how ECM and GF regulate HK motility under defined conditions: (1) in the absence of GF and ECM and (2) with or without GF with cells apposed to a known pro-motility ECM. Our results show that HK migrate on selected ECM even in the total absence of GF. This suggests that certain ECM alone are able to “initiate” HK migration. Unlike ECM, however, GF alone cannot initiate HK migration. HK cannot properly migrate when plated in the presence of GF, regardless of the concentration, without an ECM substratum. The role of GF, instead, is to augment ECM-initiated motility and provide directionality. To gain insights into the mechanism of action by ECM and GF, we compared, side-by-side, the roles of three major mitogen-activated protein kinase cascades, extracellular-signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK). Our data show that ERK1/2 is involved in mediating collagen's initiation signal and GF's augmentation signal. p38 is specific for GF's augmentation signal. JNK is uninvolved in HK motility. Constitutively activated p38 and ERK1/2 alone could not initiate HK migration. Co-expression of both constitutively activated p38 and ERK1/2, however, could partially mimic the pro-motility effects of collagen and GF. This study reveals for the first time the specific functions of ECM and GF in cell motility.