The family IV cellulose-binding domain of Clostridium thermocellum CelK (CBD_CelK) was expressed in Escherichia coli and purified. It binds to acid-swollen cellulose (ASC) and bacterial microcrystalline cellulose (BMCC) with capacities of 16.03 and 3.95 µmol/g of cellulose and relative affinities (K_r) of 2.33 and 9.87 liters/g, respectively. The CBD_CelK is the first representative of family IV CBDs to exhibit an affinity for BMCC. The CBD_CelK also binds to the soluble polysaccharides lichenin, glucomannan, and barley -glucan, which are substrates for CelK. It does not bind to xylan, galactomannan, and carboxymethyl cellulose. The CBD_CelK contains 1 mol of calcium per mol. The CBD_CelK has three thiol groups and one disulfide, reduction of which results in total loss of cellulose-binding ability. To reveal amino acid residues important for biological function of the domain and to investigate the role of calcium in the CBD_CelK four highly conserved aromatic residues (Trp₅₆, Trp₉₄, Tyr₁₁₁, and Tyr₁₃₆) and Asp₁₉₂ were mutated into alanines, giving the mutants W56A, W94A, Y111A, Y136A, and D192A. In addition 14 N-terminal amino acids were deleted, giving the CBD-N_CelK. The CBD-N_CelK and D192A retained binding parameters close to that of the intact CBD_CelK, W56A and W94A totally lost the ability to bind to cellulose, Y136A bound to both ASC and BMCC but with significantly reduced binding capacity and K_r and Y111A bound weakly to ASC and did not bind to BMCC. Mutations of the aromatic residues in the CBD_CelK led to structural changes revealed by studying solubility, circular-dichroism spectra, dimer formation, and aggregation. Calcium content was drastically decreased in D192A. The results suggest that Asp192 is in the calcium-binding site of the CBD_CelK and that calcium does not affect binding to cellulose. The 14 amino acids from the N terminus of the CBD_CelK are not important for binding. Tyr136, corresponding to Cellulomonas fimi CenC CBD_N1 Y85, located near the binding cleft, might be involved in the formation of the binding surface, while Y111, W56A, and W94A are essential for the binding process by keeping the CBD_CelK correctly folded.