Uncertainty is a fundamental feature of human life that can be fractioned into two distinct psychological constructs: risk (known probabilistic outcomes) and ambiguity (unknown probabilistic outcomes). Although risk and ambiguity are known to powerfully bias nonsocial decision-making, their influence on prosocial behavior remains largely unexplored. Here we show that ambiguity attitudes, but not risk attitudes, predict prosocial behavior: the greater an individual’s ambiguity tolerance, the more they engage in costly prosocial behaviors, both during decisions to cooperate (experiments 1 and 3) and choices to trust (experiment 2). Once the ambiguity associated with another’s actions is sufficiently resolved, this relationship between ambiguity tolerance and prosocial choice is eliminated (experiment 3). Taken together, these results provide converging evidence that attitudes toward ambiguity are a robust predictor of one’s willingness to engage in costly social behavior, which suggests a mechanism for the underlying motivations of prosocial action.