Lady Ballers satirizes contemporary discussions around gender identity and transgender women's participation in sports. Its narrative depicts cisgender men identifying as women for competitive advantage, with humor largely stemming from this premise. The overall portrayal is widely interpreted as ridiculing or denigrating transgender identities and related LGBTQ+ themes.

Lady Ballers presents a negative portrayal of transsexual themes by depicting cisgender men cynically identifying as women to win a basketball league. The film uses this premise to ridicule the concept of gender identity and the inclusion of trans women in women's sports, framing it as a source of mockery and unfair advantage.

The film, produced by a conservative media company, implicitly and explicitly champions traditional values often associated with Christianity. Its satire of modern gender ideology aligns with and affirms a traditional, often Christian, worldview regarding gender and morality, positioning characters embodying these values as reasonable.