Probably one of the most recognized but least understood phenomena of membrane transporters is the dynamic coupling between conformational change and substrate transport. In particular, the structural variability of the dynamic, non-inhibited states of transporters has been largely inaccessible by experiments and its characterization has relied almost exclusively on molecular dynamics simulation. We used nuclear magnetic resonance (NMR) approach to investigate the mitochondrial GDP/GTP carrier (GGC), a passive nucleotide transporter from yeast, in the absence and presence of substrates. NMR residual dipolar coupling (RDC) analysis of GGC in DNA nanotube liquid crystal showed that different structured segments of GGC have different generalized degree of order (GDO), indicating the presence of multiple conformations under non-inhibited conditions. Combining molecular fragment searching and RDC tensor analysis, we mapped conformational variability, as represented by change in GDO, onto the GGC structural model. Our results overall imply dynamic switching between the inward-facing and outward-facing open states of GGC in both substrate-free and substrate-bound conditions and such molecular breathing may be essential for efficient substrate transport.