Control of the parr-smolt transformation (or smoltification) is crucial for the husbandry and successful seawater (SW) transfer of Atlantic salmon (Salmo salar) reared in freshwater (FW) hatcheries. Photoperiod is an important environmental signal that initiates the complex physiological, morphological and behavioural changes that coincide with marine migration. While the use of long-day photoperiods to initiate smoltification has been well studied, this study investigated how three preparatory photoperiods in FW (LD 08:16, LD 12:12, LD 16:08) preceding exposure to 24-h light (LD 24:0) may influence or enhance smolt performance and growth post-SW transfer. After the photoperiod treatment phase (8 weeks), all groups were exposed to LD 24:0 for 8 weeks (FW) and then transferred to SW for a further 8 weeks. Exposure to LD 16:08 induced rapid development of smolt-related characteristics such as increased gill NKA activity, gill NKAα1b mRNA, and plasma cortisol, and decreased gill NKAα1a mRNA levels and condition factor through the 8-week treatment phase. Subsequent exposure to a LD 24:0 photoperiod resulted in a partial reversal of several of these characteristics, suggesting these fish went through a partial desmoltification. Exposure to LD 12:12 for 8 weeks prior to LD 24:0 elicited an intermediary response in smoltification attributes compared to LD 16:08 and LD 08:16. The LD 12:12 group adapted to SW and showed no negative effects on growth or physiological responses after transfer to SW. Exposure to a shortened photoperiod (LD 08:16) did not elicit any smoltification-related changes prior to LD 24:0, however, exposure to LD 24:0 increased gill NKA activity, plasma cortisol, changes in NKAα1a and NKAα1b mRNA, and the ratio of NKAα1b: NKAα1a. These results were confirmed by the expected changes in NKAα1a and NKAα 1b-positive immuno-reactive gill ionocytes. In summary, after exposure to LD 24:0 fish in the LD 08:16 group showed similar levels of change to those of the LD 16:08 group during the initial FW phase (prior to exposure to LD 24:0). After SW transfer, all groups were able to upregulate SW-specific NKAα1b mRNA and acclimate to SW, even though no increase in cortisol was evident. By the end of the study, there was no difference in SW growth among the groups. Overall, our data indicate that LD 16:08 advanced hypoosmoregulatory characteristics prior to LD 24:0 exposure. In addition, the physiological and molecular indicators measured in this group suggest that fish could have been transferred to SW immediately after 8 weeks in LD 16:08, with no added benefit of successive exposure to LD 24:0, which is typically used by industry to induce smoltification.