Natural polyamines play a key role in many biological processes, particularly in the stabilization of DNA double helix structure in the cell nucleus. Among others, the conformational flexibility of polyamines, such as spermidine, is an essential property for the formation of complexes with DNA. Yet, the characterization of the conformational space of polyamines has not been fully elucidated. Using atomistic molecular dynamics (MD) simulations, we present a detailed study of the conformational space of spermidine³⁺ both in solution and in interaction with DNA. We have identified more than 2000 distinct conformations, which can be grouped into seven modes. Notably, the relative population of these modes is highly affected by the interaction of spermidine³⁺ with DNA, thus representing a fingerprint of complex formation. In particular, three of the seven dihedral angles of spermidine³⁺ are predominantly in trans conformation (with or without DNA), while the other four dihedral angles are observed to switch between trans, gauche⁺ and gauche^-. The preference between the latter conformational states was analyzed in terms of the distinct energy contributions composing the potential energy. Overall, our results shed light on the conformational equilibrium and dynamics of spermidine³⁺, which in turn is important for understanding the nature of its interaction with DNA.