Night break treatment was applied to Eustoma grandiflorum 'Neilpeach Neo' using different light sources to investigate growth and flowering responses. The seedlings were grown in a greenhouse and treated with night break treatment using a far-red fluorescent lamp, a plant growth fluorescent lamp, a daylight-type fluorescent lamp or an incandescent lamp, or were grown under non-treated conditions (control). Photosynthetic photon flux density (PPFD) at the top of the plant was 5 µmol·m −2 ·s −1 under each light source. The red (660 ± 30 nm): far-red (730 ± 30 nm) ratio of the light sources was 0.01, 0.43, 5.00, and 0.65, respectively. The time from planting to flower budding was 46 days for plants grown under a far-red light fluorescent lamp, compared with 66 days in the control. The incandescent lamp and plant growth fluorescent lamp also reduced the time to flower budding; however, the daylight-type fluorescent lamp delayed flower budding by 4 days compared with the control. The light source regimes had little or no effect on the time to flowering after flower budding. The far-red fluorescent lamp, incandescent lamp and plant growth fluorescent lamp reduced the number of nodes on the main stem at the anthesis of the first floret, while the daylight-type fluorescent lamp increased the number of nodes. The farred fluorescent lamp and incandescent lamp promoted internode elongation, while the daylight-type fluorescent lamp reduced internode length. Moreover, a red fluorescent lamp with a R: FR ratio of 105 delayed flower budding and flowering, and increased the number of nodes of the plants as compared with the control, regardless of the long day lengths in July and August. Thus, it is considered that the growth and flowering time of Eustoma grandiflorum can be controlled by long-day treatment carried out with the proper type of light source.