BackgroundFemale sex workers have been disproportionately affected with HIV and anal sexual experience elevate their vulnerability. Anal intercourse has more risk of HIV transmission than vaginal intercourse for receptors that coupled with low condom and proper lubricant use behavior during anal sex. Besides majority of them did not understand HIV transmission risk of anal intercourse. In Ethiopia, studies on anal sexual experience is almost none existent, so the purpose of this study is to explored anal sexual experience and HIV transmission risk awareness among female sex worker in Dire Dawa, Eastern Ethiopia.MethodQualitative study with thematic analysis approach was conducted among 18 female sex workers and recruitment of study participants performed until saturation of information. The principal investigator conducted in-depth interviews using local language (Amharic) and it was recorded on audio recorder. Tape recorded data was transcribed and translated to English and entered into open code version 3.4 for coding and theme identification. Data collection conducted simultaneously with data analysis.ResultFemale sex workers practiced anal sex for different themes like financial influence, coercion, intentionally, peer pressure and as a sign of intimacy and love. Coercion, negative attitudes, poor awareness about HIV transmission risks of anal sex and protection capacity of condom and proper lubricants are the identified themes for not using condom and proper lubricants during anal sex by female sex workers. Inaccessibility and unavailability of health services for issues related to anal sex was the core reason for female sex workers’ misperception and risk anal sexual experience.ConclusionFemale sex workers practiced anal sex without risk reduction approaches and they did not understand exacerbated risk of anal sex to HIV transmission. Stakeholders including ministry of health need to incorporate potential awareness raising tasks and programs about risk of anal sex and methods of risk reduction for female sex workers.Electronic supplementary materialThe online version of this article (doi:10.1186/s41256-017-0047-6) contains supplementary material, which is available to authorized users.