Chilling stress generates significant inhibition of normal growth and development of cotton plants and lead to severe reduction of fiber quality and yield. Currently, little is known for the molecular mechanism of brown-fiber cotton (BFC) to respond to chilling stress. Herein, RNA-sequencing (RNA-seq)-based comparative analysis of leaves under 4°C treatment in two different-tolerant BFC cultivars, chilling-sensitive (CS) XC20 and chilling-tolerant (CT) Z1612, was performed to investigate the response mechanism. A total of 72650 unigenes were identified with eight commonly used databases. Venn diagram analysis identified 1194 differentially expressed genes (DEGs) with significant up-regulation in all comparison groups. Furthermore, enrichment analyses of COG and KEGG, as well as qRT-PCR validation, indicated that 279 genes were discovered as up-regulated DEGs (UDEGs) with constant significant increased expression in CT cultivar Z1612 groups at the dimensions of both each comparison group and treatment time, locating in the enriched pathways of signal transduction, protein and carbohydrate metabolism, and cell component. Moreover, the comprehensive analyses of gene expression, physiological index and intracellular metabolite detections, and ascorbate antioxidative metabolism measurement validated the functional contributions of these identified candidate genes and pathways to chilling stress. Together, this study for the first time report the candidate key genes and metabolic pathways responding to chilling stress in BFC, and provide the effective reference for understanding the regulatory mechanism of low temperature adaptation in cotton.