Fusarium wilt, caused by the fungus Fusarium oxysporum Schlecht. f. sp. vasinfectum (Fov) is a destructive soil-borne cotton disease. To profile the genes and pathways responding to Fov infection, we compared transcriptomic responses before and after F. oxysporum inoculation in a highly resistant cotton cultivar, Yumian21, and a highly susceptible cultivar, Jimian11. Although the overall gene expression pattern was downregulated in both cultivars, the global gene expression in the resistant cultivar was stronger than that in the susceptible cultivar. In addition, the expressed genes of two cultivars mostly differed in “cellular process,” “single-organism process,” “metabolic process,” and “response to stimulus” functional groups in the biological process Gene Ontology category: the upregulated differentially expressed genes (DEG) were largely enriched in the resistant cultivar, while the downregulated DEGs were largely enriched in the susceptible cultivar. Phenylpropanoid biosynthesis and phenylalanine metabolism are the key metabolic pathways in cotton in response to Fov. We found that lignin plays a potential role in cotton resistance to Fov. Two coding genes, caffeic acid 3-O-methyltransferase and peroxidase2, as well as the two transcription factors MYB46 and MYB86, are possibly involved in the accumulation and synthesis of lignin. Furthermore, the result showed that the quantification of lignin could be potentially used as a selection tool to identify Fusarium wilt resistant cotton.