BACKGROUND:Many gay and bisexual men (GBM) experience HIV anxiety, particularly around condomless anal intercourse. HIV pre-exposure prophylaxis (PrEP) is an effective HIV prevention strategy that may reduce HIV anxiety among GBM. METHODS:The Following Lives Undergoing Change (Flux) Study is a national, online, open-prospective observational study of licit and illicit drug use among GBM in Australia. In 2018, participants responded to newly included items regarding anxiety about HIV transmission. Stratifying GBM as high or low risk as determined by the Australian PrEP Guidelines, we assess whether PrEP use is associated with lower levels of HIV anxiety. Multivariate logistic regression was used to compare factors associated with PrEP use among GBM at high risk (PrEP-eligible) and low risk (PrEP-ineligible) of HIV infection. Results are reported as adjusted odds ratios and corresponding 95% confidence intervals. RESULTS:Among 1547 men, the mean age was 37.1 years (SD 13.1). Men aged 25 years or younger had higher HIV anxiety scores than older men. Among PrEP-eligible men, men who use PrEP reported lower levels of HIV anxiety (adjusted odds ratio = 0.92; 95% confidence interval = 0.87 to 0.99; P < 0.001). No differences were observed on HIV anxiety among PrEP-ineligible men. CONCLUSIONS:Among PrEP-eligible men, PrEP use was independently associated with lower levels of HIV anxiety. In addition to avoiding HIV infection, PrEP use may help reduce anxiety among men at risk of HIV. This feature of PrEP could be promoted as part of demand creation initiatives to increase PrEP uptake.