Serum response factor (SRF) is a highly conserved and widely expressed, single copy transcription factor that theoretically binds up to 1,216 permutations of a 10-base pair cis element known as the CArG box. SRF-binding sites were defined initially in growth-related genes. Gene inactivation or knockdown studies in species ranging from unicellular eukaryotes to mice have consistently shown loss of SRF to be incompatible with life. However, rather than being critical for proliferation and growth, these genetic studies point to a crucial role for SRF in cellular migration and normal actin cytoskeleton and contractile biology. In fact, recent genomic studies reveal nearly half of the >200 SRF target genes encoding proteins with functions related to actin dynamics, lamellipodial/filopodial formation, integrin-cytoskeletal coupling, myofibrillogenesis, and muscle contraction. SRF has therefore emerged as a dispensable transcription factor for cellular growth but an absolutely essential orchestrator of actin cytoskeleton and contractile homeostasis. This review summarizes the recent genomic and genetic analyses of CArG-SRF that support its role as an ancient, master regulator of the actin cytoskeleton and contractile machinery.