The aim of this study was to see if the crude extract of Lepidium sativum (Ls.Cr) exhibits species specificity in its antidiarrheal and antispasmodic activities along with insight into the underlying mechanisms using the in-vivo and in-vitro experiments. Ls.Cr inhibited castor oil-induced diarrhea in mice at doses (300 and 1000 mg/kg) three times higher dose than for rats. In isolated rat ileum and jejunum, Ls.Cr completely inhibited carbachol (CCh), low K+ (25 mM) and high K+ (80 mM)-induced contractions, while in guinea-pig tissues, Ls.Cr caused complete inhibition of only CCh-induced contraction. In rabbit tissues, Ls.Cr completely inhibited CCh and low K+-induced contractions sensitive to K+ channel antagonists. Pretreatment of guinea-pig and rat tissues with Ls.Cr caused a rightward shift in CCh-induced contractions in a pattern similar to dicyclomine, while in rabbit and rat tissues, Ls.Cr shifted isoprenaline curves to the left similar to papaverine. These data indicate that the antidiarrheal and antispasmodic activities of L. sativum are species dependent, mediating its antispasmodic effect through combinations of multiple pathways including activation of K+ channels, and inhibition of muscarinic receptors, Ca++ channels and PDE enzyme. Rat tissues showed the highest potency. Based on the results, we recommend using multiple species to know the real pharmacological profile of medicinal products. Copyright © 2012 John Wiley & Sons, Ltd.