The Amur ide (Leuciscus waleckii) in Lake Dali exhibits remarkable salt-alkali resistance, thriving in environments with an alkalinity of 53.57 mmol/L and pH of 9.6. It also demonstrates reproductive migration behavior, migrating annually to freshwater rivers to spawn. During this migration, the fish must face the transition of high-permeability to low-permeability water, which involves immense energy redistribution for basic metabolism and gonadal development. Steroid hormones, which are lipid hormones synthesized from cholesterol through a series of enzymatic reactions, play a crucial role in osmoregulation, gonadal development, and energy consumption during fish reproductive migration. To explore the role of steroid hormones in the osmoregulatory pressure mechanism of Leuciscus waleckii, we conducted a 10 d bidirectional cross-border stress experiment involving 50 mmol/L NaHCO₃ alkalinity stress followed by freshwater recovery. We analyzed serum steroid hormone levels, gene expression related to gill tissue in different treatment groups, and examined the methylation of key genes in promoter regions. Our results showed that differences in steroid hormones were primarily associated with three pathways. In the steroid biosynthesis pathway, the cholesterol and key genes involved (hsd17b7, ebp, dhcr24, and dhcr7) significantly increased in the alkalinity stress group (P＜0.05) and significantly decreased in the freshwater recovery group (P＜0.01). In the primary bile acid biosynthesis pathway, 25-hydroxycholesterol and 7α, 25-hydroxycholesterol, along with their target genes (ch25h and cyp7a1), significantly increased in the alkalinity stress group and significantly decreased in the freshwater recovery group (P＜0.01). In the steroid hormone biosynthesis pathway, Pregnenolone, 17α-hydroxyprogesterone, cortexolone, and cortisol significantly reduced in the alkalinity stress group (P＜0.001) and significantly increased in the freshwater recovery group (P＜0.05). Cholesterol, an intermediate product shared by these three pathways, also showed notable changes. Additionally, the promoter regions of the key genes involved in cholesterol synthesis and metabolism, ebp and sult2b1, exhibited low methylation levels in the alkalinity stress group and high methylation levels in the freshwater recovery group. These findings suggest that cholesterol is a crucial component of the steroid hormone-related pathways. It influences the methylation levels of key gene promoter regions involved in its synthesis and metabolism, thereby regulating the differential expression of other key genes in response to different environmental conditions. This regulation impacts the content of steroid hormones and plays a role in the osmoregulation of Leuciscus waleckii in varying environments. The results of this study provide a theoretical basis and scientific guidance for understanding the molecular mechanisms of fish alkali resistance, as well as for the transplantation and domestication of fish, and the cultivation of novel alkali-resistant varieties.