There is an active debate concerning the association of handedness and spatial ability. Past studies used small sample sizes. Determining the effect of handedness on spatial ability requires a large, cross-cultural sample of participants and a navigation task with real-world validity. Here, we overcome these challenges via the mobile app Sea Hero Quest. We analysed the navigation performance from 422 772 participants from 41 countries and found no reliable evidence for any difference in spatial ability between left- and right-handers across all countries. A small but growing gap in performance appears for participants over 64 years old, with left-handers outperforming right-handers. Further analysis, however, suggests that this gap is most likely due to selection bias. Overall, our study clarifies the factors associated with spatial ability and shows that left-handedness is not associated with either a benefit or a deficit in spatial ability.