Bell pepper ( Capsicum annuum L.) is a tradable and desirable crop; however, its perishable nature requires low-temperature handling. [Paradoxically](javascript:;), cold causes chilling injury (CI) and postharvest waste. Current knowledge about CI in pepper is limited. The mechanism of CI is multi-faceted; thus, this study focused on fatty acid desaturation and identified an upstream nuclear transcription factor (TF) CaMYB340 belonging to the R2R3 MYB subfamily that negatively regulates fatty acid desaturation and the ICE-CBF pathway, and is induced by low temperature (4°C) at the DNA and transcriptional levels. Specifically, McrBC treatment and bisulfite sequencing PCR indicate that exposure to cold triggers DNA methylation on one of the CHH sites in the CaMYB340 promoter. This epigenetic event at least partly contributes to the upregulation of CaMYB340 transcripts. Increased expression of CaMYB340 results in the formation of protein complexes with CaMYB340, CabHLH93, and CaMYB1R1 that in turn downregulates the expression of downstream genes. For peppers held at low temperature, transient overexpression of CaMYB340 reduced unsaturated fatty acid (FA) content and membrane fluidity, resulting in cold-induced poor peel texture. Overall, these results underscore the intricacy of transcriptional networks in plants and highlight the role of CaMYB340 in CI occurrence in pepper fruits.