Light is an important factor that influences the growth and development of flowering plants. The present study investigated the effects of in vitro acclimatization to different light colors (white light (WL; control), blue light (BL; 447 nm), green light (GL; 519 nm), and red light (RL; 667 nm)) on the growth of petunia (Petunia hybrida) and of hardening cultivation of plant transferred form in vitro to a greenhouse under sunlight. Compared to the control, the shoot length and leaf width of Petunia increased by 42% and 11.7%, respectively, after acclimatization to BL and the shoot growth increased by 29.3% after acclimatization to RL. The chlorophyll and carotenoid contents after acclimatization to BL and GL were 16.7% and 11.3% higher, respectively, and 14.4% and 11.9% higher, respectively, than those in the control. During greenhouse cultivation, the shoot length increased by 16.7% and 11.3%, respectively, after acclimatization to BL and RL, respectively, and the leaf length and leaf width increased by 14.4% and 11.9%, respectively, after acclimatization to GL. While dry weight of root of GL and BL was not significant difference in vitro, increased by 59.0% and 22.9% ex vitro than that of WL. Thus, acclimatization to BL increased the shoot growth and leaf chlorophyll contents, and acclimatization to GL and RL enhanced shoot and root growth, in petunia.