Previous studies have shown that drought occurring at anther development period will have a certain effect on pollen viability, however, it is not clear which developmental stage of cotton microspore is most sensitive to drought and the mechanism of the effect of drought during this period on pollen viability is not explored. To that end, a polyethylene glycol simulated drought experiment (Experiment 1), where drought stress was imposed at different stages of microspore development, was conducted, using cv. Yuzaomian 9110, in order to identify the most sensitive developmental stage to water deficit based on changes in microspore structure and pollen germinability. The results indicated that the microspore mother cell meiosis (MCM) stage was the most sensitive to water deficit. According to that, a second study (Experiment 2) was conducted, using the same cultivar, and the effects of drought stress during MCM-stage on pollen’s carbohydrate and energy metabolism were monitored during and after the stress and their influences on pollen viability were revealed. As per our results, MCM-stage drought down-regulated the sucrose transporter gene GhSUT9 expression which inhibited sucrose import to the anthers; however, increases in sucrose phosphate synthase activity with concurrent decreases in sucrose synthase (SuSy) and invertase (INV) activities resulted in high sucrose content in the water-stressed anthers. In spite of the decreased sucrose decomposition, anther hexose contents were higher in the water-stressed anthers than well-watered anthers, probably due to a decrease in hexose oxidation through the tricarboxylic acid cycle as it was later confirmed by the reduced anther adenosine triphosphate (ATP) content under drought. Contrastingly to sucrose and hexose, anther starch content was greatly reduced by drought, due to decreases in the activities of starch synthesizing enzymes, ADP-glucose pyrophosphorylase (AGPase) and soluble starch synthase (SSSase). After the relief of stress, anther sucrose content decreased to levels lower than well-watered anthers, due to unrecovered GhSUT9 expression and increased alkaline INV activity which enhanced sucrose decomposition. Despite the increased fructose content, glucose content decreased to levels lower than well-watered anthers, without however, resulting in higher energy supply as it was indicated by the lower pollen ATP content after the relief of stress. Anther starch remained lower than well-watered anthers, even after the stress was relieved, as starch biosynthesizing enzymes, AGPase and SSSase, remained suppressed. All these findings suggest that drought stress occurring at the MCM-stage has the most detrimental effect to cotton’s pollen viability due to alterations in carbohydrate metabolism that result in inadequate energy supply.