Objectives/purposes of the study: Vulvodynia is common and characterized by vulvar discomfort and pain. Yet few studies have assessed hygienic practices in relation to onset. We investigated whether hygienic behaviors were associated with the onset of vulvodynia. Methods: We assessed self-reported history of personal hygienic behaviors, including wearing tight fitting clothing, vulva care and genital washing, pubic hair removal, douching, and powdering, a year prior to first reported onset of vulvar pain among 213 clinically confirmed cases and a similar time period among 221 general population controls. Results: Compared to women who reported never wearing tight fitting jeans or pants, women wearing tight fitting jeans or pants ≥4 times per week had twice the odds of vulvodynia (95%CI: 1.14-3.95). Relative to controls, women with vulvodynia were substantially less likely to report use of soaps and gels to cleanse the vulva (95%CI 0.17-0.63). Among women who chose to remove pubic hair, those who removed pubic hair from the mons pubis compared to bikini-area only hair removal, were 74% more likely to have vulvodynia (95%CI 1.05-2.89). Finally, compared to women who reported bikini-area only hair removal less than monthly, those who removed hair from the mons pubis weekly or more were nearly 2 times more likely to be vulvodynia cases (95%CI 0.83-3.49). Conclusions: Wearing tight fitting jeans or pants and removing hair from the mons pubis area were associated with increased odds of vulvodynia. Research on how hygienic practices could influence vulvar pain in larger and more temporally addressed populations is warranted. Precis Wearing tight fitting jeans or pants and removing hair from the mons pubis were associated with an increased odds of clinically-confirmed vulvodynia.