A previous study detected mixing of two deeply split mtDNA clades (Clade I and Clade II) for Atlantic and Mediterranean populations of the medusozoan Pelagia noctiluca. The north hemisphere glaciations and the Messinian salinity crisis have been proposed as the two main biogeographic events related to the isolation between the Atlantic Ocean and the Mediterranean Sea. We tested if the splitting time between Clade I and Clade II of P. noctiluca was associated with one of these geological events. Our study was based on DNA sequence data of mitochondrial (COI and 16S ribosomal RNA) and nuclear (18S ribosomal RNA, internal transcribed spacer 1 and 5.8S ribosomal RNA) genes from populations of the Atlantic and Pacific Ocean and the Mediterranean Sea. The rise of the Isthmus of Panama was used to calibrate substitution rates for COI. This calibration was based on the detection of a shallow but significant genetic structure between P. noctiluca populations from the Pacific and the Atlantic Oceans. Considering our calibration for COI, we refute a possible origin of Clades I and II during the Messinian salinity crisis. Our estimates suggest the origin for a putative common ancestor of Clades I and II around 2.57 Ma (with 95% 2.91–2.22 HPD), roughly corresponding to the Gelasian stage of the early Pleistocene. These alterations include changes in the sea level and oceanic currents at the Strait of Gibraltar and other regions of the Mediterranean basin, and could explain the origin of the two P. noctiluca clades.