Molecular tweezers based on chenodeoxycholic acid bearing different lengths of arm were synthesized andtheir anion binding affinities were evaluated by $^1H$ NMR, isothermal calorimetric titration, and ESI mass spectrometry. Molecular tweezer 6 showed a high selectivity toward $H_2PO_4;^-$ over $Cl^-,;Br^-,;I^-, $ and $CH_3CO_2;^-$ by $^1H$ NMR titration, whereas the association constant for $F^-$ revealed the largest value as determined by ITC. The selectivity of 6 towards $F^-$ was about 103 times higher than that of $Cl^-,;H_2PO_4;^- $, and $CH_3CO_2;^-$. ITCexperiment of 6 with $F^-$ in a DMSO showed two binding modes; two sequential association constants $K_1;=;2.77;{ imes};10^5;M^{-1}$ and $K_2;=;8.68;{ imes};10^6;M^{-1}$ were found. These sequential bindings were confirmed by ESI massspectrometry. 1 : 1 and 1 : 2 complexes of 6 and $F^-$ were found at m/z 868.08 and 884.04.