There are sharp U.S. political differences in transgender attitudes, with liberals expressing much more favorable attitudes and support for trans-inclusive policies than conservatives. Transgender attitudes likely contribute to numerous public policies that are being passed or considered, including policies that impact transgender health, safety, and well-being. Thus, it is imperative to understand why vast partisan differences in transgender attitudes occur. Here we present two preregistered studies examining whether transgender knowledge (i.e., knowledge about transgender identities, experiences, causes, and health care) differs in Democrats versus Republicans, and whether knowledge mediates political differences in attitudes and policy support (N = 439, Prolific). Participants completed a transgender quiz, followed by measures of transgender prejudice and support for trans-inclusive policies. Democrats had superior transgender knowledge compared to Republicans (ds > 1.39). Further, as predicted, transgender knowledge significantly mediated political differences in transgender prejudice and policy support. Exploratory analyses found that the predicted mediation effects held when knowledge was operationalized in different ways and after adjusting for demographic covariates. These data suggest that knowledge gaps help to explain political differences in transgender attitudes and may stimulate additional work on how to improve such knowledge.