The role of calcium in mediating resistance to several auxinic herbicides (i.e., 2,4-dichlorophenoxyacetic acid, [4-chloro-2-methylphenoxy] acetic acid, (±)-2-(4-chloro-2-methylphenoxy) propanoic acid [mecoprop], 3,6-dichloro-2-methoxy-benzoic acid [dicamba], or 4-amino-3, 5,6-trichloro-2-pyridinecarboxylic acid [picloram]) was investigated by modulating calcium dynamics of a susceptible (S) and resistant (R) biotype of wild mustard. The inhibitory effects of the auxinic herbicides on root length of the S seedlings were significantly reduced upon pretreatment with calcium in the presence of the calcium ionophore A23187. Conversely, the addition of verapamil, a calcium channel blocker, to the R seedlings increased their sensitivity to the auxinic herbicides. Valinomycin, a potassium channel ionophore, did not ameliorate the effect of the auxinic herbicides on both biotypes of wild mustard, thus indicating that the observed effects were specific for calcium. These results demonstrate that calcium plays a crucial role in the resistance of wild mustard to auxinic herbicides at the level of intact seedlings, thereby supporting our previous results using intact protoplasts.