Although natalizumab (anti-α4 integrin) represents an effective therapy for relapsing remitting multiple sclerosis (RRMS), it is associated with an increased risk of developing progressive multifocal leukoencephalopathy (PML), caused by the polyomavirus JC (JCV). The aim of this study was to explore natalizumab-induced phenotypic changes in peripheral blood T-lymphocytes and their relationship with JCV reactivation. Forty-four patients affected by RRMS were enrolled. Blood and urine samples were classified according to natalizumab infusion number: 0 (N0), 1-12 (N12), 13-24 (N24), 25-36 (N36), and over 36 (N > 36) infusions. JCV-DNA was detected in plasma and urine. T-lymphocyte phenotype was evaluated with flow cytometry. JCV serostatus was assessed. Ten healthy donors (HD), whose ages and sexes matched with the RRMS patients of the N0 group, were enrolled. CD8 effector (CD8 E) percentages were increased in natalizumab treated patients with detectable JCV-DNA in plasma or urine compared to JCV-DNA negative patients (JCV-) (p < 0.01 and p < 0.001, resp.). Patients with CD8 E percentages above 10.4% tended to show detectable JCV-DNA in plasma and/or urine (ROC curve p = 0.001). The CD8 E was increased when JCV-DNA was detectable in plasma or urine, independently from JCV serology, for N12 and N24 groups (p < 0.01). As long as PML can affect RRMS patients under natalizumab treatment with a negative JCV serology, the assessment of CD8 E could help in the evaluation of JCV reactivation.