RWBY features multiple LGBTQ+ characters, most notably the central romantic relationship between Blake Belladonna and Yang Xiao Long, which is depicted as a source of strength and affirmation. Other queer characters, such as Ilia Amitola and May Marigold, are also portrayed with dignity and respect, contributing to a consistently positive and validating representation within the narrative.

RWBY features May Marigold, a transgender character portrayed positively as a competent and brave member of the Happy Huntresses. Her backstory highlights finding acceptance and chosen family after facing external prejudice, affirming the worth of her identity. The series depicts her with dignity and agency, without her identity being a source of ridicule or villainy.

The show frequently portrays female characters achieving victory in direct physical combat against male opponents. Examples include Yang Xiao Long's tournament win against Mercury Black, Pyrrha Nikos's dominant performance against a team of male students, and Neo Politan's swift defeat of Roman Torchwick.