The 130kDa smooth muscle myosin light chain kinase (smMLCK) is a Ca²⁺/calmodulin-regulated enzyme that plays a pivotal role in the initiation of smooth muscle contraction and regulating cellular migration and division. Despite the critical importance of the smMLCK in these processes, little is known about the mechanisms regulating its' expression. In this study we identified the proximal promoter of smMLCK within an intron of the mouse MYLK gene. The MYLK gene encodes at least two isoforms of MLCK (130kDa and 220kDa) and telokin. Luciferase reporter gene assays demonstrated that a 282bp fragment (-167 to +115) of the smMLCK promoter was sufficient for maximum activity in A10 smooth muscle cells and 10T1/2 fibroblasts. Deletion of the 16 base pairs between -167 and -151, which included a CArG box, resulted in a nearly complete loss of promoter activity. Gel mobility shift assays and chromatin immunoprecipitation assays demonstrated that serum response factor (SRF) binds to this CArG box both in vitro and in vivo. SRF knockdown by shRNA decreased endogenous smMLCK expression in A10 cells. Although the SRF coactivator myocardin induced smMLCK expression in 10T1/2 cells, myocardin only activated the promoter 2-4 fold in reporter gene assays. Addition of intron 1 or 6kb of 5' upstream sequence did not lead to any further activation of the promoter by myocardin. The proximal smMLCK promoter also contains a consensus GATA binding site that bound GATA-6. GATA-6 binding to this site decreased endogenous smMLCK expression, inhibited promoter activity in smooth muscle cells and blocked the ability of myocardin to induce smMLCK expression. Together these data suggest that SRF and SRF-associated factors play a key role in regulating expression of the smMLCK.