S100A4 is a member of the S100 family of proteins and increases the motility of many cell types. This is also thought to explain its association with the epithelial-mesenchymal transition (EMT), a developmental program re-activated during tumourigenesis. Mechanistically, S100A4 interacts with a number of targets including Smad3 and liprin-β1; however, the best characterised is non-muscle myosin IIA (NMIIA) which regulates many aspects of the cytoskeleton. There is a large body of in vitro data indicating that S100A4 promotes the monomeric state of NMIIA; however, in vivo evidence for the interaction in cells is lacking. Accordingly, the first aim of this study was to determine if S100A4 interacts with, and promotes the monomeric state of NMIIA in A431 cells undergoing SIP1-induced EMT. Intriguingly, co-localisation analysis of S100A4 and NMIIA in A431-SIP1 cells using immunoelectron microscopy indicated that NMIIA is present in a folded, 10S state, and unfolded 6S state, and S100A4 interacts with both. This represents the first evidence of 10S and 6S states of NMIIA in non-muscle cells. In addition, FRAP analysis demonstrated that cells with attenuated expression of S100A4 turned over NMIIA with a slower rate, consistent with S100A4 promoting the monomeric state. The second part of the study explored the mechanism of the S100A4-NMMIA interaction. In vitro analysis of phosphomimetic S1916D and S1943D NMIIA showed no differences in binding affinity with S100A4 compared to WT NMIIA, contrary to the published literature. Based on the NMR structure of S100A4 and NMIIA, V77 and C81 were identified as key S100A4 residues that mediated the interaction with NMIIA. Mutation of these sites abolished the interaction with NMIIA, an effect reflected in null-phenotypes for both proteins when over-expressed in A431 cells compared to WT S100A4. In conclusion, this study suggests S100A4 is an important regulator of NMIIA dynamics in cells.