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<i>CONSTANS</i> (<i>CO</i>) is an important regulator of photoperiodic flowering and functions at a key position in the flowering regulatory network. Here, two <i>CO</i> homologs, <i>MiCOL16A</i> and <i>MiCOL16B</i>, were isolated from “SiJiMi” mango to elucidate the mechanisms controlling mango flowering. The <i>MiCOL16A</i> and <i>MiCOL16B</i> genes were highly expressed in the leaves and expressed at low levels in the buds and flowers. The expression levels of <i>MiCOL16A</i> and <i>MiCOL16B</i> increased during the flowering induction period but decreased during the flower organ development and flowering periods. The <i>MiCOL16A</i> gene was expressed in accordance with the circadian rhythm, and <i>MiCOL16B</i> expression was affected by diurnal variation, albeit not regularly. Both the MiCOL16A and MiCOL16B proteins were localized in the nucleus of cells and exerted transcriptional activity through their MR domains in yeast. Overexpression of both the <i>MiCOL16A</i> and <i>MiCOL16B</i> genes significantly repressed flowering in <i>Arabidopsis</i> under short-day (SD) and long-day (LD) conditions because they repressed the expression of <i>AtFT</i> and <i>AtSOC1</i>. This research also revealed that overexpression of <i>MiCOL16A</i> and <i>MiCOL16B</i> improved the salt and drought tolerance of <i>Arabidopsis</i>, conferring longer roots and higher survival rates to overexpression lines under drought and salt stress. Together, our results demonstrated that <i>MiCOL16A</i> and <i>MiCOL16B</i> not only regulate flowering but also play a role in the abiotic stress response in mango.