The CONSTANS-like (COL) family plays a pivotal role in regulating plant photoperiodic flowering pathways. Although several COLs have been characterized in Arabidopsis, their functions in cotton lack clarity. Here, GhCOL3, a gene of the COL family in cotton (Gossypium hirsutum), was cloned and characterized. GhCOL3 is located in the nucleus, and GhCOL3 was expressed in young leaves, hypocotyls, and flower organs and exhibited obvious circadian rhythms under long-day conditions. Overexpressing of GhCOL3 heterogeneously in Arabidopsis thaliana led to delayed flowering, whereas silencing of GhCOL3 in cotton using the virus-induced gene silencing system led to earlier flowering, suggesting a negative regulatory role of GhCOL3 in plant flowering. Transcriptome analysis and expression detection showed that bHLH38, bHLH100, bHLH101, and BBX31 were significantly upregulated in GhCOL3 heterogeneous overexpression lines, whereas the expression of FT was downregulated. Moreover, the expression of GhbHLH38, GhBBX31, and GhFT were significantly affected in the GhCOL3-silenced line, thus laying the foundation for elucidating the regulatory mechanism of GhCOL3 in cotton flowering.