Herein we report a series of [LnL(H2O)]Cl3 complexes, in which L is a 1,4,7,10-tetrazacyclododecane framework (cyclen) functionalized with 2-methylpyridyl pendants on the nitrogen atoms at the 1- and 7-positions and with N-[2-(2-hydroxyethoxy)ethyl]acetamide groups on those at the 4- and 10-positions, and Ln stands for Eu, La, and Tb. The complexes were isolated and characterized by elemental analysis, mass spectrometry, infrared spectroscopy, and 1H NMR spectroscopy for the La and Eu complexes. 1H NMR spectroscopy indicates that the complexes display a major twisted square antiprismatic (TSAP) geometry in solution, which was confirmed by using DFT calculations. 1H and 13C variable-temperature NMR spectroscopic experiments on the La complex provided an activation free energy of ΔG≠ = (58 ± 3) kJ mol–1 for the enantiomerization process of the TSAP isomer, which requires both the inversion of the five-membered chelate rings formed upon coordination of the cyclen moiety and the rotation of the pendant arms. The photophysical properties of the complexes were studied by means of absorption, steady-state, and time-resolved luminescence spectroscopy. Upon ligand excitation, the Eu and Tb complexes displayed the typical lanthanide-centered emission spectra with weak emission efficiencies (Φov = 0.11 and 0.68 %, respectively, for Eu and Tb in H2O). Luminescence lifetime measurements in water and heavy water allowed the determination of the hydration number of the complexes; these were (1.0 ± 0.2) for Eu and Tb. Spectrophotometric titrations of the [LnL(H2O)]Cl3 complexes (Ln = Eu and Tb) with F–, Cl–, and Br– anions (in buffered water solutions, 0.01 M tris(hydroxymethyl)aminomethane (TRIS)/HClO4, pH = 7.0) showed the complexes to interact selectively with fluoride anions, whereas no interactions were observed with Cl– and Br–. The interaction with F– was monitored by luminescence spectroscopy with [EuL(H2O)], which evidenced dramatic changes in the shape of the luminescence spectra, and an association constant of (2.86 ± 0.06) log units was determined. In the case of the Eu complex, time-resolved and high-resolution emission spectroscopy showed that the interaction resulted in the displacement of the coordinated water molecule, which led to an increase of the overall luminescence quantum yield, with almost no impact on the ligand-to-metal sensitization efficiency. Investigation of the Eu and La complexes by potentiometry (0.1 M NaCl) allowed the determination of the protonation constants of the complexes as well as the association constants with fluoride anions. DFT calculations on the fluoride adduct of the Eu complex confirmed the higher stability of the TSAP isomer and evidenced large structural changes upon F– binding, in agreement with spectrophotometric observations in solution.