Mammary gland health plays a key role in maintaining lactation persistency. As a well-known factor involved in physiological processes, the role of oxygen levels in bovine mammary health and lactation persistency remains to be investigated. The present study aimed at investigating the potential regulatory role of hypoxia in the mammary gland of dairy cows with different lactation persistency. Sixty-one Holstein dairy cows were selected for a 180-day experiment at approximately 88 days in milk (DIM). Plasma, milk and mammary tissue samples from 61 cattle were collected on experimental days 0, 90 and 180 (corresponding to 88, 178 and 268 DIM), respectively. Of the 61 cows, 12 cows with high lactation persistency (HP) and 12 with low lactation persistency (LP) were selected for the current study. No difference was observed in milk yield between two groups on d 0 (Pd 0 = 0.67), whereas differences emerged between animals with different lactation persistency at d 105 (Pd 105 = 0.03) until d 180 (Pd 180 < 0.01). The level of mammary apoptosis was significantly higher in the LP group than in the HP cows (Ppersistency < 0.01). In the oxygen-related variables, plasma concentration of hypoxia-inducible factor 1α (HIF-1α) was higher in the LP cows than in the HP group (Ppersistency < 0.01), especially on d 0 (Pd 0 < 0.01). Compared with HP cows, LP cows had a higher malonaldehyde (Pd 180 = 0.01) and a lower activity of inducible nitric oxide synthase (Pd 180 = 0.01) on d 180, suggesting a possible oxygen alteration between cows with different lactation persistency. RNA-sequencing analysis of the mammary gland on d 0 revealed that HIF-1 associated molecules may play a role in driving mammary gland apoptosis in dairy cows. A lower lactation persistency of dairy cows may be resulted from the altered HIF-1α in the mammary gland.