Calmodulin (CaM) regulates numerous cellular functions by sensing Ca2+ levels inside cells. Although its structure as a function of the Ca2+-bound state remains hotly debated, no report is available on how pH independently or in interaction with Ca2+ ions regulates shape and function of CaM. From SAXS data analysis of CaM at different levels of Ca2+-ion concentration and buffer pH, we found that (1) CaM molecules possess a Gaussian-chain-like shape in solution even in the presence of Ca2+ ion or at low pH, (2) the global shape of apo CaM is very similar to its NMR structure rather than the crystal structures, (3) about 16 Ca2+ ions or more are required per CaM molecule in solution to achieve the four-Ca2+-bound crystal structure, (4) low pH alone can impart shape changes in CaM similar to Ca2+ ions, and (5) at different [Ca2+]/[CaM] ratio or pH values, the predominant shape of CaM is essentially a weighted average of its apo and fully activated shape. Results were further substantiated by analysis of sedimentation coefficient values from analytical ultracentrifugation and peptide binding assays using two peptides, each known to preferentially bind the apo or the Ca2+-activated state.