The bifunctional mesoporous zirconium phosphonates (ZrBFs) were synthesized through surfactant-assisted co-condensation of ZrCl4 with two different phosphonic acids, both 1-phosphomethylproline (H3PMP) and 1,4-bis(phosphomethyl)-piperazine (BPMP), in a one-pot procedure. The L-proline group of H3PMP and piperazine group of BPMP in the frameworks endow ZrBFs with pH-controllable release function and high cell penetration capability, which was derived from the reversible protonation-deprotonation of L-proline groups and piperazine groups on the mesoporous walls under different pH values (pH sensitivity) as well as further functionalization with biological modifiers via the carboxyls in L-proline groups on the outer surface (functionalizability), respectively. ZrBFs, possessing cationic frameworks once formed, exhibit high payload for salmon sperm DNA as model nucleic acid owing to strong electrostatic attraction between them. On the basis of pH-sensitive ZrBFs carriers and assisted by lag-time films coating, the time- and pH-controlled oral colon-targeted nucleic acid delivery systems have been developed, which can carry most of the loaded salmon sperm DNA to the colon under dual control, time control and pH value control. Furthermore, salmon sperm DNA can remain intact during delivery, as evidenced by the fact that the released salmon sperm DNA in the pH transition release experiment still retain its structural integrity and native conformation. Also, fluorescence spectra demonstrate that ZrBFs can be further functionalized with a cell-penetrating peptide of octaarginine (R8) via the carboxyls in L-proline groups of H3PMP on the outer surface using a coupling agent, which will enhance the penetration capability of ZrBFs through biomembranes. ZrBFs have a potential application as a new kind of carrier in oral delivery of nucleic acids targeting the colon for gene therapy of colon-related diseases due to their unique bifunctionality.