HIV-positive men who have sex with men (MSM) have an increased risk for anal human papillomavirus (HPV) infection, anal high-grade intraepithelial lesions (HSIL), and anal cancer. Smoking is associated with abnormal anal cytology and with an increased risk for anal cancer. We collected 3736 intraanal swabs from 803 HIV-positive MSM who participated in an anal cancer screening program between October 2003 and August 2014. HPV prevalence, anal cytology and HPV DNA load of high-risk (HR) HPV-types 16, 18, 31 and 33 of non-smokers and smokers were compared. HPV-typing was performed by alpha-HPV genus-specific PCR and hybridization with 38 type-specific probes using a multiplex genotyping assay. In samples positive for HPV16, 18, 31, or 33, HPV DNA loads were determined by type-specific real-time PCRs and expressed as HPV DNA copies per betaglobin gene copy. At baseline, HR-HPV DNA (80.5 vs. 89.0%, p=0.001), HPV16 DNA (41.6 vs. 52.3%, p=0.003), HPV18 DNA (15.5 vs. 26.0%, p<0.001), anal dysplasia (LSIL+HSIL; 51.5 vs. 58.4%, p=0.045) and HSIL (17.2 vs. 22.7%, p=0.048) were detected more frequently in smokers compared to non-smokers. Throughout the study period 32.7% of non-smokers and 39.9% of smokers developed HSIL (p=0.011), and three smokers developed anal cancer. Considering swabs from the entire study period (median HPV load value per patient per cytology grade), smokers with normal anal cytology had significantly higher HPV16 loads (median 0.29 vs. 0.87, n=201, p=0.007) and cumulative high-risk-HPV loads (median 0.53 vs. 1.08, n=297, p=0.004) than non-smokers. Since elevated HR-HPV DNA loads are associated with an increased risk for HPV-induced anogenital cancers, HPV-infected HIV-positive MSM should be counseled to refrain from smoking. Additionally, for smokers, shorter anal cancer screening intervals than for non-smokers may be appropriate.