The pigment components in green cotton fibers were isolated and identified as 22-O-caffeoyl-22-hydroxymonodocosanoin and 22-O-caffeoyl-22-hydroxydocosanoic acid. The concentration of 22-O-caffeoyl-22-hydroxymonodocosanoin was correlated positively with the degree of green fibers of color, indicating a role of caffeoyl derivatives in pigmentation of green cotton fibers. Upland cotton (Gossypium hirsutum L.) contains four genes (Gh4CL1 to Gh4CL4) encoding 4-coumarate:CoA ligase (4CL), key enzymes of the phenylpropanoid biosynthesis pathway. In 15 to 24 days-post anthesis fibers, the expression level of Gh4CL1 was very low, Gh4CL3 had a similar expression level between the white and green cottons, Gh4CL2 had a significantly higher expression level in the green fibers than in the white fibers, whereas Gh4CL4 seemed to have a higher expression level in the white fibers than in the green fibers. According to enzyme kinetics analysis, Gh4CL1 displayed a preference for 4-coumarate, Gh4CL3 and Gh4CL4 exhibited a somewhat low but still prominent activity towards ferulate, while Gh4CL2 had a strong preference for caffeate and ferulate. These results suggest that Gh4CL2 might be involved in the metabolism of caffeoyl residues and related to pigment biosynthesis in green cotton fibers. Our findings provide insights for understanding biochemical and molecular mechanisms of pigmentation in green cotton fibers.