The concept of a mild mutagen was coined to describe a minor mutagenic activity exhibited by some nucleoside analogues that potentiated their efficacy as antiretroviral agents. In the present study, we report the mild mutagen activity of sofosbuvir (SOF) for hepatitis C virus (HCV). Serial passages of HCV in human hepatoma cells, in the presence of SOF at a concentration well below its cytotoxic concentration 50 (CC ) led to pre-extinction populations whose mutant spectra exhibited a significant increase of C→U transitions, relative to populations passaged in the absence of SOF. This was reflected in an increase in several diversity indices that were used to characterize viral quasispecies. The mild mutagenic activity of SOF was largely absent when it was tested with isogenic HCV populations that displayed high replicative fitness. Thus, SOF can act as a mild mutagen for HCV, depending on HCV fitness. Possible mechanisms by which the SOF mutagenic activity may contribute to its antiviral efficacy are discussed.