Cotton (Gossypium hirsuturm L.) vacuolar H+-ATPase plays an important role in fiber cell elongation through regulating the turgor pressure, which is involved in polarity enlargement of fiber cell. Arabidopsis vacuolar H+-ATPase subunit C (DET3) regulates the activity of vacuolar H+-ATPase and subsequently promotes cell elongation. To rapidly characterize the function of cotton vacuolar H+-ATPase subunit C (GhDET3) and speculate the role of GhDET3 in cotton fiber growth and development, we constructed an expressing vector for fission yeast and introduced it into fission yeast. The results revealed that overexpression of GhDET3 was capable of promoting fission yeast cell elongation and enhancing the tolerance to high NaCl and high pH stresses. These results illuminated that the effects of GhDET3 were important for the activity of vacuolar H+-ATPase and suggested that GhDET3 plays a crucial role in fiber cell elongation.