An investigation was made on the strain-controlled low-cycle fatigue (LCF) of K40S cobalt-base superalloy at 900 °C in ambient atmosphere. The results show that K40S alloy possesses high LCF resistance in comparison with X-40 alloy. Under the testing conditions in this study, K40S alloy exhibits a cyclic stress response of initial hardening followed by softening. The cyclic stress response behavior has been attributed to dislocation-dislocation interactions and dislocation-precipitate interactions. The high response stress can lead to a large stress concentration at locations where inelastic strains of high amplitude accumulate, which account for the decreasing fatigue life with increasing strain rate. The well-distributed carbide particles are the “secondary” crack initiation sites. The secondary crack initiation relaxes the stress concentration at the crack tip, reducing the driving force of crack propagation. High-temperature LCF failure of K40S alloy results from the interaction of the mechanical fatigue and environmental oxidation.