Measurement of pKa values for model derivatives demonstrated that the 3,3-diFOx fragment is a weak acceptor that can be used to fine-tune acidity/basicity by replacing either donor alkyl groups or stronger acceptors like CF3, CO2Me, or C(O)NH2. This was confirmed by linear free energy relationship studies, with the Hammet constant value of the title substituent ( = 0.24) being close to that of chlorine ( = 0.29). Lipophilicity measurements for a series of model derivatives showed that the 3,3-diFOx moiety slightly increases the compound's lipophilicity when introduced into the parent molecule (LogP = +0.5). Again, this effect was intermediate between those of (cyclo)alkyl groups and polar fragments like amide, ester, 1,3dioxolane, or non-fluorinated oxetane, thus suggesting potential for isosteric replacements of both these categories. In terms of three-dimensional structure, the 3,3-diFOx fragment showed intermediate ring puckering between oxetane and cyclobutane/gem-difluorocyclobutane and was the second smallest between these fragments. The molecular electrostatic potential (MEP) map of the 3,3-diFOx group was X-shaped, with opposite potential signs at the diagonal ring atoms. As a result, the overall dipole moment of 3,3-difluorooxetane was relatively low (0.39 D), suggesting that the proposed moiety can be a better mimic for (cyclo)alkyl groups than simpler gem-difluorinated or oxygen-containing counterparts. Finally, the 3,3-diFOx moiety had reasonable stability towards common chemical reagents, excellent shelf storage stability (over 5 years), and good metabolic stability. The latter fact was confirmed by introducing the title functional group into the molecule of Tenovin-6, a known sirtuin inhibitor, instead of a tertbutyl group. This modification retained a significant fraction of the compound's biological activity, increased hydrophilicity, and reduced P-glycoprotein affinity. These data confirm bioisosteric replacement compatibility of the 3,3-diFOx moiety for the particular case studied. We hope that along with all other results presented in this work, they will promote applications of 3,3difluorooxetane as a promising functional group in drug discovery and other areas of science.