Climate change is leading to rapid change in aquatic environments, increasing the mean and variability of temperatures, and increasing the incidence of hypoxia. We investigated how acclimation to constant temperatures or to diel temperature fluctuations affects hypoxia tolerance in mummichog killifish (Fundulus heteroclitus). Killifish were acclimated to constant cool (15°C), constant warm (25°C) or a diel temperature cycle (15°C at night, 25°C during day) for 6 weeks. We then measured hypoxia tolerance (time to loss of equilibrium in severe hypoxia, tLOE; critical O2 tension, Pcrit), whole-animal metabolism, gill morphology, haematology and tissue metabolites at 15°C and 25°C in a full factorial design. Among constant temperature groups, tLOE was highest and Pcrit was lowest in fish tested at their acclimation temperature. Warm-acclimated fish had lower metabolic rate at 25°C and greater gill surface area (less coverage of lamellae by interlamellar cell mass, ILCM), but cool-acclimated fish had greater brain glycogen stores. Therefore, effects of constant temperature acclimation on hypoxia tolerance were temperature specific and not exhibited broadly across test temperatures, and they were associated with different underlying mechanisms. Hypoxia tolerance was less sensitive to test temperature in fish acclimated to fluctuating temperatures compared with fish acclimated to constant temperature. Acclimation to fluctuating temperatures also increased haemoglobin–O2 affinity of the blood (decreased P50) compared with constant temperature groups. Therefore, acclimation to fluctuating temperatures helps maintain hypoxia tolerance across a broader range of temperatures, and leads to some distinct physiological adjustments that are not exhibited by fish acclimated to constant temperatures.