We evaluated the acceptability of the 25 mg dapivirine vaginal ring (DVR) as an HIV prevention intervention and its influence on DVR adherence in the MTN-020/ASPIRE phase III trial. Acceptability measures were captured using ACASI at month 3 and end of product use (median 24 months, IQR 15-30). Monthly returned rings were classified as nonadherent if dapivirine release rate was ≤ 0.9 mg/month. Associations between acceptability measures and nonadherence were estimated using Poisson regression models with robust standard errors. At month 3 (N = 2334), 88% reported DVR was comfortable, 80% were unaware of it during daily activities, and 74% never felt it during sex. At exit, 66% were 'very likely' to use DVR in the future. Acceptability was found to differ significantly by country across several measures including wearing the ring during sex, during menses, partner acceptability, impact on sexual pleasure and willingness to use the ring in the future. Risk of nonadherence at month 12 was elevated if DVR was felt during sex at month 3 (aRR 1.67, 95% CI 1.26, 2.23). Risk of nonadherence in the last year of study participation was elevated if, at exit, participants minded wearing during sex (aRR 2.08, 95% CI 1.52, 2.85), during menses (aRR 1.57, 95% CI 1.06, 2.32), reported a problematic change to the vaginal environment (aRR 1.57, 95% CI 1.12, 2.21), and were not "very likely" to use DVR in the future (aRR 1.31, 95% CI 1.02, 1.68). DVR acceptability was overall high yet varied by country. Addressing perceived ring interference with sex, menses, or problematic changes to the vaginal environment in future interventions could help improve adherence, as could embracing sex-positive messaging related to ring use and increased pleasure.Trial Registration ClinicalTrials.gov Identifier: NCT01617096.