Calmodulin (CaM) is a universal regulatory protein that modulates numerous cellular processes by using calcium (Ca) as the signal. In smooth muscle cells (SMC), one major target of CaM is myosin light chain kinase (MLCK), a kinase that phosphorylates the myosin regulatory light chain and thereby regulates cell contraction. In the absence of CaM, MLCK remains inhibited by its autoinhibitory domain (AID). While it is well established that CaM activates MLCK, the molecular interactions between these two proteins remain elusive due to the lack of structural data. In this work, we constructed a molecular model of mammalian CaM (mCaM) in complex with MLCK leveraging AlphaFold, published biochemical data, and protein-protein docking. The model, along with a strategic set of CaM mutants including a inhibitory variant soybean CaM isoform 4 (sCaM-4), was subject to molecular dynamics (MD) simulations. Using principal component analysis (PCA), we mapped out the transition path for the removal of the AID from the MLCK kinase domain to provide molecular basis of MLCK activation. Additionally we established MLCK conformations that correspond to the active and inactive states of the kinase. We showed that mCaM and sCaM-4 cause MLCK to undergo the transition to the active and inactive states, respectively. Using two structural metrics, we computed the probabilities of MLCK activation by different CaM variants, which were in good agreement with the experimental data. Distributions along these metrics revealed that different inhibitory CaM variants impair MLCK activation through unique mechanisms. We finally identified molecular contacts that contribute to the MLCK activation by CaM. Overall, we report a molecular model of CaM-MLCK that provides insights into the molecular mechanism of MLCK activation by CaM. The mechanism requires effective removal of the AID while preserving an active configuration of the kinase domain. This mechanism may be shared by other MLCK isoforms and potentially other structurally similar kinases with CaM-mediated regulatory domains.