The effects of intrinsic structural flexibility of calmodulin protein on the mechanism of its allosteric conformational transition are investigated in this article. Using a novel in silico approach, the conformational transition pathways of intact calmodulin as well as the isolated N- and C-terminal domains are identified and energetically characterized. It is observed that the central alpha-helix linker amplifies the structural flexibility of intact Ca2+-free calmodulin, which might facilitate the transition of the two domains. As a result, the global conformational transition of Ca2+-free calmodulin is initiated by the barrierless transition of two domains and proceeds through the barrier associated unwinding and bending of the central alpha-helix linker. The binding of Ca2+ cations to calmodulin further increases the structural flexibility of the C-terminal domain and results in a downhill transition pathway of which all regions transit in a concerted manner. On the other hand, the separation of the N- and C-terminal domains from calmodulin protein loses the mediating function of central alpha-helix linker, leading to more difficult conformational transitions of both domains. The present study provides novel insights into the correlation of the integrity of protein, the structural flexibility, and the mechanism of conformational transition of proteinlike calmodulin.