This article presents the controlled synthesis of a rare example of C,C′-linked bis-cyclam architecture in mild conditions through the “bis-aminal” route previously used for the advantageous synthesis of cyclam, N- and C-functional cyclams and N,N′-bis-cyclams. Two synthetic pathways were explored with the smart design of α,β-unsaturated ketones or alkyl halides bis-cyclizing agents. The first led to the isolation of a key intermediate for the future design of N-functionalized bis-cyclams, whereas the second allowed the preparation of the targeted C,C′-xylylene-bis-cyclam under mild conditions with decent yield. This compound was then studied as a CXCR4 receptor inhibitor, one of the main applications known for bis-macrocyclic compounds, in particular in the context of HIV (human immunodeficiency virus) infection. Although results demonstrated that its potency is lower (i.e. 137-fold higher IC50) than the gold standard AMD3100 against HIV infection, clear evidence of CXCR4 inhibition is presented, confirming the potential of this novel architecture and related compounds in this research field.