Miniature rose plantlets at the flower development stage were grown photo-autotrophically on MS medium and subsequently exposed to water deficits of –0.23, –0.32, –0.40, or –0.67 MPa osmotic potential (Ψs) for 14 d. The Ψs in the culture medium was raised by increasing the concentration of mannitol, which caused abnormal floral development in terms of the flowering percentage and the number of flowers per plantlet, as well as delayed flowering. In vitro flowering and the number of flowers per plantlet declined significantly when miniature rose plantlets were exposed to water deficit stress at –0.40 MPa or –0.67 MPa. Reductions in growth, pigment degradation, chlorophyll a fluorescence, and net photosynthetic rate (Pn) were greatest in plantlets exposed to a water deficit stress of –0.67 MPa. This was particularly evident in the case of Pn, with a decline of 73.7% compared to non-stressed control plantlets. In contrast, proline levels increased in plantlets under water deficit stress, as proline performs a key role as an osmoprotectant under such conditions. The flowering stage in miniature rose plantlets is particularly susceptible to water deficit stress, which suppresses the development of reproductive organs. Knowledge of the responses to water deficit stress at the reproductive stage may be applied to identify effective indices for the selection of genotypes with increased tolerance to water deficit in miniature rose breeding programmes.