Soil waterlogging and high temperature conditions generally occur together, especially in the Yangtze River Valley, China, negatively affecting cotton (Gossypium hirsutum L.) fibre development. Therefore, combined elevated temperature (34.1/29.0°C) and soil waterlogging (6 days) were imposed to study their combined effects on fibre biomass and fibre qualities (length, strength and micronaire). The results showed that in the boll cohort exposed to waterlogging and/or elevated air temperature, combined elevated temperature and soil waterlogging decreased final fibre length (by 8.9-11.3%) and fibre biomass (by 25.8-33.9%) more than either stress individually. A total of 113, 263 and 290 differential abundance proteins were identified related to elevated temperature, waterlogging and the two treatments combined, respectively, in fibres at 15 days after anthesis via the isobaric tags for relative and absolute quantitation technique, which were classified as: carbohydrate and energy metabolism (21.7%), protein metabolism (16.6%), amino acid metabolism (12.8%), intracellular structural components (6.6%), transport (7.9%), oxidation-reduction process (7.9%), signal transduction (5.2%), lipid metabolism (5.2%), stress response (5.2%), nucleic acid metabolism (4.5%), organic acid metabolism (3.4%) and others (2.1%). Both vacuolar ATPase (V-ATPase) and plasma membrane H+-ATPase (PMH+-ATPase) were responsible for fibre length formation, although V-ATPase expression may play a major role in determining fibre cell elongation rather than PM H+-ATPase expression. It was concluded that fibre cell elongation and secondary wall thickening were inhibited mainly by reduced accumulation of osmolytes, blocked synthesis and transport of secondary wall components, and disruption of the cytoskeleton system under combined elevated temperature and soil waterlogging.