© 2018 D’Amario et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. To understand the response of marine calcifying organisms under high CO2 scenarios, it is critical to study their calcification patterns in the natural environment. This paper focuses on a major calcifying phytoplankton group, the coccolithophores, through the analysis of water samples collected along a W-E Mediterranean transect during two research cruises, in April 2011 (Meteor cruise M84/3) and May 2013 (MedSeA cruise 2013). The Mediterranean Sea is a marginal sea characterized by large biogeochemical gradients. Currently, it is undergoing both warming and ocean acidification, processes which are rapidly modifying species distribution and calcification. The species Emiliania huxleyi largely dominates the total coccolithophore production in present day oceans and marine basins, including the Mediterranean Sea. A series of morphometric measurements were performed on the coccoliths of this species to estimate their mass, length and calculate a calcification index (proxy for the size-normalized calcification degree). The most abundant morphotype of E. huxleyi in the Mediterranean Sea is Type A. Coccoliths of this morphotype were additionally analyzed based on scanning electron microscopy images: four calcification varieties were quantified, according to the relationship between slit length-tube width, and the state of the central area (open or closed). The average E. huxleyi coccolith mass along the Mediterranean oceanographic transect depended strongly on both the average coccolith length and calcification index. The variability in average coccolith length and calcification index across samples reflected oscillations in the relative abundance of the calcification varieties. We also demonstrated that the distribution of the calcification varieties followed the main environmental gradients (carbonate chemistry, salinity, temperature, nutrient concentrations). Hence, shifts in the distribution of the calcification varieties and of the average E. huxleyi coccolith mass are to be expected in the Mediterranean Sea under climate change. These physiological and ecological responses will modulate the net coccolithophore calcification and, ultimately, the regional carbonate export to the seafloor.