Damage of the mucosal barrier in HIV infection, microbial translocation, and immune activation can persist even in patients on successful antiretroviral therapy (ART) especially advanced late presenters. The aim of this study was to find factors that determine immune activation and bacterial translocation in HIV-infected advanced late presenters on suppressive ART. Forty-three late presenters (CD4 < 200 cells/µl prior to ART) on successful ART (more than 2 years of ART) with optimal and suboptimal CD4 recovery were enrolled into this study. The serum concentrations of intestinal fatty acid-binding peptide (I-FABP), zonulin-1, programmed cell death-1 protein (PCDP-1), and soluble (s)CD14 were measured using the ELISA test. We found higher serum levels of I-FABP and sCD14 in successfully antiretroviral-treated advanced late presenters compared to healthy subjects (p < 0.0001 and p = 0.0004). The serum concentration of PCDP-1 and zonulin-1 in HIV-infected patients did not differ from healthy controls. The levels of microbial translocation and immune activation markers were not associated with the degree of CD4 recovery. A serum concentration of I-FABP above 2.03 ng/ml was independently associated with a shorter ART (OR 0.78; p = 0.03). Older age was related to serum levels of sCD14 above 2.35 µg/ml (OR 1.1; p = 0.01). Higher serum levels of I-FABP and sCD14 in successfully antiretroviral-treated advanced late presenters compared to healthy subjects suggest an incomplete reconstruction of the intestinal barrier and sustained immune activation despite good CD4 recovery. It was not the CD4 level, but the length of the suppressive ART that was found to be associated with the restoration of the intestinal barrier.