OBJECTIVE: To evaluate the knowledge and acceptability of the human papillomavirus (HPV) vaccine among health professionals from western Amazonia. METHODS: A cross-sectional study was conducted in the Sistema Assistencial è Saúde da Mulher e da Criança (Health Care System for Women and Children; SASMC) in Acre, Brazil, in 2017. The participants comprised 196 health professionals. The data collection instrument contained 31 questions about HPV, its clinical repercussions for women, and the HPV vaccine. Quantitative variables were presented as medians and 95% confidence intervals (CIs), and p<0.05 was considered statistically significant. For the analyses, chi-square tests and Mann-Whitney tests were used. The collected data were analyzed using Stata®11.0. RESULTS: Of the 196 health professionals, 39.8% (n=76) were physicians and 61.2% (n=120) were other health professionals. The interviewees were mostly female (n=143, 73%, 95% CI 66.1 to 78.9%) who worked in the medical field (n=81, 41.3%, 95% CI 34.4 to 48.6%), and the median age was 38 years (95% CI 36.0 to 39.7). Physicians had increased knowledge regarding only the statement "cervical cancer is one of the main causes of cancer in women", with a proportion ratio of 0.88 (0.80; 0.97) and p<0.001. Regarding clinical knowledge of the HPV vaccine, a low proportion of correct answers was obtained for all the questions, and no significant differences were found between the groups. CONCLUSION: Acceptability and knowledge of HPV and its vaccine were similar among health professionals, with knowledge gaps in questions about the relation between smoking and cervical cancer and specific clinical knowledge.