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Cotton fiber yield depends on the density of fiber cell initials that form on the ovule epidermis. Fiber initiation is triggered by MYB-MIXTA-like transcription factors (GhMMLs) and requires a sucrose supply. Ethylene or its precursor ACC (1-aminocyclopropane-1-carboxylic acid) is suggested to affect fiber yield. The <i>Gossypium hirsutum</i> (L.) genome contains 35 <i>ACS</i> genes (<i>GhACS</i>) encoding ACC synthases. Here, we explored the role of a GhACS family member in the regulation of fiber initiation. Expression analyses showed that the <i>GhACS6.3</i> gene pair was specifically expressed in the ovules during fiber initiation (3 days before anthesis to 5 days post anthesis, −3 to 5 DPA), especially at −3 DPA, whereas other <i>GhACS</i> genes were expressed at very low or undetectable levels. The expression profile of <i>GhACS6.3</i> during fiber initial development was confirmed by qRT-PCR analysis. Transgenic lines overexpressing <i>GhACS6.3</i> (<i>GhACS6.3</i>-OE) showed increased ACC accumulation in ovules, which promoted the formation of fiber initials and fiber yield components. This was accompanied by increased transcript levels of <i>GhMML3</i> and increased transcript levels of genes encoding sucrose transporters and sucrose synthase. These findings imply that GhACS6.3 activation is required for fiber initial development. Our results lay the foundation for further research on increasing cotton fiber production.