Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and the varied outcomes of the infection depend on both viral and host factors. We have demonstrated that the HCV alternate reading frame protein (F protein) is related to Th1/Th2 bias which is involved in virus persistence in chronic hepatitis C (CHC) patients. The purpose of this study was to test the hypothesis that genetic variants of TBX21 (T cell specific T-box transcription factor) were associated with the outcomes of HCV infection and F protein generation. Three single nucleotide polymorphisms (SNPs) (rs17250932, rs2074190, rs4794067) in the TBX21 gene were genotyped in a case-control study in a cohort of a high-risk group, including 354 healthy controls and 747 CHC patients (190 anti-F protein antibody seronegative patients and 557 anti-F protein antibody seropositive patients). Results showed that the rs4794067 C allele in the TBX21 promoter was significantly more common in CHC patients (OR = 1.335, 95% CI = 1.058-1.684, P = 0.015), exceptionally in anti-F protein seropositive patients (OR = 1.547, 95% CI = 1.140-2.101, P = 0.005), compared with healthy controls. And the risk effect was also significantly high in patients with HCV 1b genotype and mild fibrosis (P = 0.021, P = 0.010, respectively). Compared with the most frequent haplotype TAT, haplotype analysis showed that the distribution of TAC was significantly different between the chronic HCV carrier group and the healthy group, and so was the anti-F antibody seronegativity group and the anti-F antibody seronegativity group (all P < 0.001). Our results suggested that TBX21 variants may be involved in the etiology of this disease.