The F-box protein family is characterized by an F-box motif that has been shown to play an important role in regulating various developmental processes and stress responses. In this study, a novel F-box-containing gene was isolated from leaves of pepper cultivar P70 (Capsicum annuum L.) and designated CaF-box. The full-length cDNA is 2088 bp and contains an open reading frame of 1914 bp encoding a putative polypeptide of 638 amino acids with a mass of 67.8 kDa. CaF-box was expressed predominantly in stems and seeds, and the transcript was markedly upregulated in response to cold stress, abscisic acid (ABA) and salicylic acid (SA) treatment, and downregulated under osmotic and heavy metal stress. CaF-box expression was dramatically affected by salt stress, and was rapidly increased for the first hour, then sharply decreased thereafter. In order to further assess the role of CaF-box in the defense response to abiotic stress, a loss-of-function experiment in pepper plants was performed using a virus-induced gene silencing (VIGS) technique. Measurement of thiobarbituric acid reactive substances (TBARS) and electrolyte leakage revealed stronger lipid peroxidation and cell death in the CaF-box-silenced plants than in control plants, suggesting CaF-box plays an important role in regulating the defense response to abiotic stress resistance in pepper plants.