Field data, U-Th dating and amino acid racemisation (AAR) and epimerisation (IAE) analyses were performed in a sector of the Ionian coast of Apulia. Palaeoshoreline 1 (PS1) was identified at +40 ± 5 m with the associated terraced deposit 1 (TD1). Palaeoshoreline 2 (PS2) was identified at +30 ± 2 m with the associated terraced deposit 2 (TD2), characterised by five sub-units. The sub-units that mark the transgression have an age of 127.851 ± 1.47 ky BP (by U-Th dating) and 128.4 ± 26.2 ky BP (by AAR); one of sub-units marking the highstand falls within aminozone E (MIS 5.5) based on IAE. Palaeoshoreline 3 (PS3) was identified at +19 ± 2 m with the associated terraced deposit 3 (TD3); they mark the occurrence of a second, lower elevation MIS 5.5 highstand. TD3 dates back to aminozones Eu2013D (MIS 5.5-MIS 5.3) based on IAE and to 122.49 ± 1.25 ky BP by U-Th dating. We considered two scenarios where the first highstand of MIS 5.5 occurred at 125 and 123 ky BP, with a sea level at +7.5 ± 1.5 m; we then calculated the uplift rate of PS2 for both scenarios: it resulted to be 0.18 ± 0.03 mm/year. These uplift rates were used to calculate the position of PS3 at 122.49 ± 1.25 (rounded to u2053122 ky BP) as well as the position of the layer dated to 127.851 ± 1.47 ky BP (rounded to u2053128 ky BP). The position of PS3 at the time of its formation (i.e., the sea level at u2053122 ky BP, corresponding to the second highstand of MIS 5.5) was calculated to be −2.96 ± 5.42 m for Scenario 1 and −3.32 ± 5.47 m for Scenario 2. The sea level obtained by the layer dated to ~128 ky BP was found to have a value of −11.79 ± 6.29 m. Finally, we reconstructed a relative sea level (RSL) trend suggesting two main highstands during MIS 5.5, with the earlier higher than the second.