Natural killer (NK) cells constitute a first line of defense against viral infections; their function is governed by the integration of signals from multiple activating and inhibitory surface receptors. We hypothesized that because NKs become rapidly activated by cytokines, response to anti‐hepatitis C virus (HCV) therapy would be predicted by the phenotype and function of NKs. We used a cohort of 101 patients (55 African, 46 Caucasian‐American) who received pegylated‐interferon (IFN) and ribavirin for 48 weeks. Multiparameter FACS analysis was used to examine relative expression of 14 different inhibitory/activating receptors. Interleukin (IL)‐28B genotyping (rs12979860) was also performed. Pretreatment levels of inhibitory receptors CD158a, CD158b, and CD158e were higher in patients who demonstrated poor viral decline within the first 28 days of therapy. Higher expression levels of inhibitory receptors NKG2A, CD158b, and CD158e were demonstrable in patients who failed to achieve sustained virologic response (SVR). Patients carrying the IL‐28B T allele had higher NKG2A expression on effector NKs. We created a mathematical regression model incorporating race, viral level, and two inhibitory receptors. The area‐under‐the curve was 0.88, which is highly predictive of SVR. Moreover, the model performed complementarily with IL‐28B across the CC, CT, and TT genotypes. Purified NKG2A^neg NKs treated with pegylated‐IFN‐α for 4 hours demonstrated higher levels of IFN‐γ‐inducible protein‐10 (IP‐10) and tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) compared with their NKG2A^pos counterparts. Conclusions: These results provide novel insights into the associations of NK phenotype with IL‐28B genotype and gene expression patterns, as well as the role of NKs in mediating IFN‐induced viral clearance of chronic HCV infection. (HEPATOLOGY 2011;)