Treatment of chronic hepatitis C virus (HCV) infection with direct‐acting antivirals results in a rapid decline in viral load and markers of hepatic inflammation, including serum chemokine (C‐X‐C motif) ligand 10 (CXCL10) concentration, which is followed in most cases by a sustained virologic response. Whether parallel changes of significance occur in the cellular composition of peripheral blood is relatively unknown. We hypothesized that longitudinal characterization of peripheral blood during treatment would provide insight into cellular migration and immune activation, which would have implications for understanding host immunity both before and after HCV treatment and may relate to HCV clearance. We analyzed longitudinal peripheral innate and adaptive immune cell populations by flow cytometry from 95 subjects enrolled in two direct‐acting antiviral clinical trials and examined chemokine receptor expression on T lymphocytes in 43 patients. Within 1‐2 weeks of initiating treatment, significant increases were observed in the concentration of peripheral cluster of differentiation 4–positive (CD4⁺) and CD8⁺ T lymphocytes but not monocyte or natural killer cells. In tandem with these changes, the percent of both CD4⁺ and CD8⁺ T lymphocytes with an activated phenotype (human leukocyte antigen [HLA] DR⁺ and CD38⁺) decreased, and T‐lymphocyte surface expression of chemokine (C‐X‐C motif) receptor 3, the chemokine receptor for CXCL10, increased. Conclusion: Rapid changes in peripheral cellular populations occur during direct‐acting antiviral treatment of HCV infection, which could potentially relate to hepatic efflux of tissue lymphocytes due to altered inflammation and chemokine receptor signaling, providing critical insight into the relationship between host immunity and viral clearance during HCV infection. (Hepatology Communications 2017;1:586–594)