The G protein-coupled chemokine receptor CXCR4 is implicated in a variety of physiological responses that also share several downstream effectors involved in multiple pathological processes. The interaction between CXCR4 and its natural ligand CXCL12/stromal-derived factor-1 (SDF-1) plays important roles in cancer metastasis, HIV-1 infection, and inflammatory diseases. Therefore, investigating the CXCR4-CXCL12 interaction is critical for understanding the molecular mechanisms of the modulation of chemokine-receptor functions and designing new pharmaceutical agents to target the CXCR4-CXCL12 pathway. Based on known experimental data, the interaction between CXCR4 and CXCL12 was predicted by an integrated protocol, which combines protein-protein docking, molecular dynamics (MD) simulations, Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) binding free energy calculations, and MM/GBSA binding free energy decomposition analysis. The predicted CXCR4-CXCL12 binding pattern is in good agreement with the experimental data. Analysis of the binding structure reveals an obvious electrostatic complementarity between CXCR4 and CXCL12. Moreover, significant conformational rearrangements were observed during the 50 ns MD simulations. In particular, the basic Lys1 at the CXCL12 N-terminus, an essential residue in receptor activation, forms a strong polar interaction with the Glu32 in the CXCR4 extracellular region. It facilitates the significant movement of TM5 and TM6 in the conformational transition, which is coupled to the association with the intracellular signal transduction pathways via heterotrimer G-protein. Based on the dynamic and energetic analyses, a two-site binding model was proposed. We believe that our study provides useful information for understanding the mechanisms of CXCR4 ligand binding and structure-based drug design of CXCR4.