Calbindin D28k is a highly conserved Ca2+-binding protein abundant in brain and sensory neurons. The 261-residue protein contains six EF-hands packed into one globular domain. In this study, we have reconstituted calbindin D28k from two fragments containing three EF-hands each (residues 1–132 and 133–261, respectively), and from other combinations of small and large fragments. Complex formation is studied by ion-exchange and size-exclusion chromatography, electrophoresis, surface plasmon resonance, as well as circular dichroism (CD), fluorescence, and NMR spectroscopy. Similar chromatographic behavior to the native protein is observed for reconstituted complexes formed by mixing different sets of complementary fragments, produced by introducing a cut between EF-hands 1, 2, 3, or 4. The C-terminal half (residues 133–261) appears to have a lower intrinsic stability compared to the N-terminal half (residues 1–132). In the presence of Ca2+, NMR spectroscopy reveals a high degree of structural similarity between the intact protein and the protein reconstituted from the 1–132 and 133–261 fragments. The affinity between these two fragments is 2 × 107 M−1, with association and dissociation rate constants of 2.7 × 104 M−1 s−1 and 1.4 × 10−3 s−1, respectively. The complex formed in the presence of Ca2+ is remarkably stable towards unfolding by urea and heat. Both the complex and intact protein display cold and heat denaturation, although residual α-helical structure is seen in the urea denatured state at high temperature. In the absence of Ca2+, the fragments do not recombine to yield a complex resembling the intact apo protein. Thus, calbindin D28k is an example of a protein that can only be reconstituted in the presence of bound ligand. The α-helical CD signal is increased by 26% after addition of Ca2+ to each half of the protein. This suggests that Ca2+-induced folding of the fragments is important for successful reconstitution of calbindin D28k.