A series of p-tert-butyldihomooxacalix[4]arenes bearing phenolic hydroxyls and thiourea moieties were prepared to investigate their anion binding behavior. These compounds showed F⁻ binding according to ¹H NMR titration experiments. Among them, dihomooxacalix[4]arenes bearing phenyl and benzyl thiourea (2a and 2b) showed multiple interactions with added F⁻, including N–H⋯F, O–H⋯F hydrogen bonds and “through-space” C–H⋯F⁻ interactions between F⁻ and the attached aromatic segment of thiourea. A conformational change of the dihomooxacalix[4]arene cavity was observed as well and proved by theoretical calculations. The dihomooxacalix[4]arene bearing methyl thiourea (2c) also showed the formation of N–H⋯F, O–H⋯F hydrogen bonds, but less conformational change of the dihomooxacalix[4]arene cavity according to ¹H NMR titration results. The bis-dihomooxacalix[4]arene (3) showed clear N–H⋯F hydrogen bond formation upon addition of F⁻ as characterized by ¹H NMR spectroscopy.