Atmospheric CO2 levels have been increasing at an accelerated rate for the last 250 years, much of which is absorbed by the oceans, resulting in a process called ocean acidification (OA). This phenomenon has the capacity to disrupt many marine biological processes that utilise carbon, in particular photosynthesis and calcification, and as such phytoplankton have been a main topic of OA studies. While research has accelerated over the last decade, establishing general trends still remains confounded by methodological inconsistencies. Coccolithophores, particularly the species Emiliania huxleyi, are both ecologically and biogeochemically important phytoplankton; however, one strain (NZEH) has produced highly varied results. Here, we present a multivariate analysis that suggests previous inconsistencies between past studies of NZEH may be driven by variance of the light:dark (L:D) cycle used for growth. Experimental analysis on NZEH showed that under a 14:10h L:D cycle, CO2 induces significantly slower growth rates and higher PIC and POC cell-1, but this effect is dampened under 24h of light. This was widened to encompass more taxa, including more isolates of E. huxleyi (PLY70-3, PLY124-3, RCC962), and two other species of coccolithophore; Gephyrocapsa oceanica and Coccolithus pelagicus. L:D cycle changed the observed OA response, with two main responses divided by biogeographical origin. In tropical taxa, 24h light enhanced the effects of increased photosynthesis, but dampened the decrease in calcification in response to CO2. For temperate taxa, 24h dampened both the increases in photosynthesis and calcification with CO2. Evaluation of photobiology reveals that both CO2 and longer photoperiods induce a “high light” acclimation response, and changes in coccosphere thickness suggest it has a photoprotective role. Finally, results from bioassay experiments on natural phytoplankton populations in the polar regions show that CO2 response is hard to predict and based on community composition and ambient starting conditions. This work serves to further highlight the importance of environmental variables that moderate the OA response in accurately understanding future biogeochemical cycles. Future models attempting to predict the impact of OA upon marine systems must critically account for interactive role of light availability.