MKL1 (MRTF-A/MAL) is a member of the myocardin-related transcription factor family that plays a key role in the development and differentiation of smooth muscle cells (SMCs) via activation of serum response factor (SRF)-dependent SMC gene expression. MKL1 associates with SRF and stimulates its transcriptional activity. Here, by performing matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis combined with in vitro glutathione S-transferase pull-down assay, we identified 4 candidate proteins that associate with MKL1 through the N-terminus region of MKL1. SPT16, ATP citrate lyase, nucleolin and radixin were identified, and the physical and functional interactions between MKL1 and SPT16 were examined. SPT16 is a component of the FACT (facilitating chromatin transcription) complex that allows RNA polymerase II to traverse the nucleosomes. SPT16 associates with MKL1 in vitro and in vivo; moreover, SSRP1, another component of the FACT complex, associates with the N-terminus region of MKL1 in vitro. SPT16 synergistically activates the transcriptional activity of MKL1. These results show that the expression of nucleosomal SRF-dependent genes, including the SMC gene, is activated by MKL1 via activation of SRF and recruitment of the FACT complex.