Behavioural abnormality in fishes has been proposed as a significant threat of the increasing levels of carbon dioxide occurring in the oceans. Negative effects of elevated CO2 have been reported for behaviours such as predator-prey interactions, foraging, hearing and behavioural lateralisation. Importantly, the effects vary greatly both within and between species, and some recent studies have shown minimal effects of CO2 on behaviour. Whether the effect of CO2 also varies between males and females is however virtually unexplored. According to resource allocation theory, females are expected to be more sensitive to elevated CO2, meaning that non-sex specific studies may overlook ecologically important differences between the sexes. In this study, we investigated the possible differences between males and females in their response to elevated CO2 by performing behavioural lateralisation tests in adult temperate two-spotted gobies Gobiusculus flavescens. We found that the strength of the side bias (absolute lateralisation) was unaffected by the CO2 treatment, and there was no difference between males and females. The control fish were slightly right-biased in their behavioural asymmetry (mean relative lateralisation of 14). Exposure to high CO2 affected this pattern, such that treated fish were slightly left biased (mean relative lateralisation of -10), regardless of their sex. The same results were obtained yet again when the study was repeated during a second year. We discuss our results in light of the great variation in lateralisation that has been reported to depend on variables such as species, ecological settings and environmental factors.