Under a variety of circumstances p38α Mitogen-activated Protein Kinase (p38α) is activated by an unusual mechanism that is independent of upstream kinases and involves autophosphorylation of the TGY motif within the activation loop. One such mechanism is initiated by TAB1, a scaffold protein. By co-expression in mammalian, bacterial and cell-free systems we show that TAB1 is able to induce p38α autophosphorylation. We identify the region on TAB1 responsible for p38 recognition and activation. A chemically synthesized peptide spanning this region recapitulates the biophysical and biological behaviour of full-length TAB1 in vitro and in vivo. We are able to show that the chemically synthesized peptide increases the affinity of p38α towards ATP and as a consequence p38α autophosphorylates its activation loop in cis, moreover the autoactivation of p38α through TAB1 interaction can be artificially induced in adult ventricular rat myocytes and in whole hearts using the synthetic peptide. Finally through mutagenesis analysis, NMR spectroscopy and X-ray crystallography we are able to describe the molecular determinants that underlie the mechanism of auto-activation in p38. Taken together these data shed light on the molecular mechanism through which p38α is able to autophosphorylate in cis. Interference with this mechanism may allow circumstance specific inhibition of p38α activation. Potentially, such intervention could circumvent the drawbacks seen when pharmacological inhibitors of p38 catalytic activity are used clinically.