A combination of humidity-dependent single crystal to single crystal (SC−SC) structural transformation and single crystal proton conductivity measurements is essential to elucidate the underlying proton transport mechanism in metal−organic framework materials. Herein, we report a new layered Co−Ca phosphonate [Co III Ca II (notpH 2 )(H 2 O) 2 ]ClO 4 •nH 2 O [abbreviated as CoCa•nH 2 O, where notpH 6 = 1,4,7-triazacyclononane-1,4,7-triyl-tris-(methylenephosphonic acid), C 9 H 18 N 3 (PO 3 H 2 ) 3 ]. CoCa•nH 2 O undergoes a reversible relative humidity (RH) dependent SC−SC structural transformation between CoCa•2H 2 O and CoCa• 4H 2 O at room temperature. Accordingly the continuous hydrogen bond network observed in CoCa, leading to a drastic decrease in proton conductivity by ∼5 orders of magnitude. The process is reversible; hence, the proton conductivity is tunable simply through humidity control. The AC impedance measurements using single crystals of CoCa•nH 2 O reveal that the [010] direction of H-bond extension is the preferred proton conduction pathway showing the greatest conductivity of 1.00 × 10 −3 S cm −1 at 25 °C and 95% RH. Although the [20−1] direction, which involves the phosphonate oxygen atoms in the H-bond network shows the lowest conductivity of 4.35 × 10 −8 S cm −1 at 25 °C and 95% RH, the ClO 4 − anions play a key role in not only connecting the lattice water molecules into a continuous hydrogen bond network but also assisting the proton diffusion between the lattice water molecules. This work provides a rare example of a proton conductive MOF with a well-illustrated proton conduction mechanism and is a promising humidity sensor for future applications.