Pegylated interferon (Peg-IFN)-α2a resulted in viral suppression and reduction in integrated proviral HIV DNA in 9 of 20 antiretroviral therapy (ART)-suppressed subjects undergoing analytical ART interruption (ATI; NCT00594880). To confirm the first pilot study, here we evaluated if Peg-IFN-α2b would be safe, maintain viral suppression during ATI, and decrease latent viral reservoir in chronic HIV infection. 20 individuals with well controlled HIV infection (on ART, VL <50 copies/ml) received weekly 1 μg/kg Peg-IFN-α2b sc for 20 weeks, with a 4 week ATI (weeks 5-9 of IFN treatment). In addition to safety monitoring, several HIV measures (integrated HIV DNA, TILDA, rectal tissue measures, etc.) were assessed at baseline and week 20. Final statistical analysis: we used Wilcoxon Signed rank test to test differences between time points; mixed effect models and hierarchical clustering to test relationships between HIV reservoir measurements. At completion study participants were 20% females, 70% AA. Median age was 47. 18 subjects completed treatment (2 early terminations) with 7 serious events (neutropenia). Peg-IFN-α2b suppressed plasma HIV RNA during the 4 week ATI in 52% (95% CI= 32-73%), higher than historical controls (13%; 95% CI= 3-36%, p= 0.0127; NCT00051818). At week 20, we observed a significant reduction in HIV RNA-expressing GALT cells (p= 0.012) and a reduction in integrated HIV DNA in circulating CD4s (p= 0.0797). A higher baseline level of rectal mucosa RNA, integrated DNA, TILDA, p24 and 2LTR were associated with a greater decrease after the intervention (p<0.05). Overall, treatment with Peg-IFN-α2b (20 weeks, 4-week ATI) 1) is safe and well tolerated, 2) maintains viral suppression during a 4-week ATI in half of the subjects, and 3) is associated with significant decrease of rectal mucosa HIV RNA. The latter supports that Peg-IFN-α2 may be an HIV cure-directed strategy to reduce HIV beyond ART.