Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the nature's inventory of sex hormones effects.