Axonemal and cytoplasmic dyneins share a highly conserved 8 kDa light chain (DLC8) for motor assembly and function. Other than serving as a light chain of dynein complexes, DLC8 has been shown to bind a larger number of proteins with diverse biological functions including cell cycle control, apoptosis, and cell polarity maintenance. Therefore, DLC8 is likely a multifunctional regulatory protein. DLC8 exists as a dimer in solution, and the protein dimer is capable of binding to two target molecules. In this work, the backbone dynamics of DLC8, both in its apo- and target-peptide bound forms, were characterized by 15N NMR relaxation studies. The relaxation data were analyzed using model-free approach. We show that the target peptide-binding region of apo-DLC8 experiences microsecond-to-millisecond time scale conformational fluctuation, suggesting that the target-binding region of the protein is capable of adjusting its shape and size in responding to its various targets. The conformational breathing of the target-binding region of apo-DLC8 was also supported by backbone amide exchange experiment. Such segmental conformational motion of the protein is significantly reduced upon forming a complex with a target peptide. The dynamic properties of DLC8 in solution provide insight into the protein's diverse sequence-dependent target binding.