The cell-microenvironment interaction is critical for cells to perceive environmental cues and co-ordinate signaling cascades to regulate physiological functions. Herein, a soft-lithography technique, micropattern via micromolding in capillaries (MIMIC), is explored to create cell-adhesive micropatterns on glass coverslips. Genetically encoded Src and RhoA fluorescence resonance energy transfer (FRET) biosensors are used to monitor the Src and RhoA activities in nonpatterned cells (stochastically migrating cells, SMCs) and those constrained to grow on micropatterned surfaces (restrictedly migrating cells, RMCs). The results reveal that epidermal growth factor (EGF) induces a decrease of RhoA and an increase of Src activities with biphasic time courses in RMCs. In contrast, the time courses of such activities in SMCs upon EGF stimulation are relatively monophasic. The inhibition of Src activity, actin network, or myosin machinery abolishes the biphasic RhoA response upon EGF stimulation in RMCs. The results indicate that this microenvironment effect on the biphasic RhoA activation in RMCs is mediated by Src and actomyosin machinery. Through the integration of FRET and micropatterning technologies, it is demonstrated that the microenvironment impacts significantly on cell shapes and subsequently the spatiotemporal signaling network of RhoA and Src in living cells. The results help to advance mechanistic understanding of how cells perceive and interpret microenvironments to co-ordinate intracellular molecular signals and ultimately physiological responses.