Upland cotton (Gossypium hirsutum L.) shows strong heterosis. However, heterosis is not widely utilized owing to the high cost of hybrid seed production. Creation of a photoperiod-sensitive genetic male sterile line could substantially reduce the cost of hybrid seed production in upland cotton. Such a mutant with virescent marker was found by space mutation in near-earth orbit and its traits had been stable after 4 years of selection in Anyang and Sanya, China. This mutant was fertile with an 11–12.5 h photoperiod when the temperature was higher than 21.5 °C and was sterile with a 13–14.5 h photoperiod. Genetic analysis indicated that both traits were controlled by a single recessive gene or two closely linked genes. Also, the cytological observations and transcriptome profiling analysis showed that the degradation of pollen grain cytoplasm should be the primary reason why the mutant line were male sterile under long-day conditions.