Astronomically tuned cyclic sedimentary successions provide unprecedented insight into the temporal evolution of depositional systems and major geologic events. However, placing astronomically calibrated records into an absolute time frame with confidence requires independent and precise geochronologic constraints. Astronomical tuning of the precessionally modulated sedimentary cycles of the Mediterranean Basin deposited during the Messinian Salinity Crisis (5.96–5.33 Ma) has indicated an ∼90 k.y. “Messinian gap”, corresponding to the evaporative drawdown of the Mediterranean following the closure of the Mediterranean-Atlantic gateway. In the Messinian deposits, a volcanic ash dated by 40Ar/39Ar geochronology was used to anchor the sedimentary cycles to the insolation curve. However, the uncertainty of the 40Ar/39Ar date introduces a potential two-cycle (∼40 k.y.) uncertainty in the tuning. Using high-precision chemical abrasion–thermal ionization mass spectrometry (CA-TIMS) U-Pb geochronology on single zircon grains from two Messinian ash layers in Italy, we obtained dates of 5.5320 ± 0.0046 Ma and 5.5320 ± 0.0074 Ma with sub-precessional resolution. Combined with our astronomical tuning of the Messinian Lower Evaporites, the results refine the duration of the “Messinian gap” to at most 28 or 58 ± 9.6 k.y., which correlates with either the TG12 glacial interval alone, or both TG12 and TG14 glacial intervals, supporting the hypothesis of a glacio-eustatic contribution in fully isolating the Mediterranean from the Atlantic Ocean. Our new U-Pb dates also allow us to infer a precessionally modulated cyclicity for the post-evaporitic deposits, and hence enable us to tune those successions to the insolation curve.