Previous studies have unravelled glycolytic oscillations in cancer cells, such as HeLa cervical and DU145 prostate cancer cells, using a monolayer culture system. Here, we demonstrate glycolytic oscillations in HeLa cervical cancer cell spheroids. Experiments revealed that a small number of HeLa cells in spheroids exhibited heterogeneous oscillations with a higher frequency than those in monolayers. Model analyses and our previous experiments indicated that the higher frequencies of oscillations in spheroids were mostly due to the increase in glycolytic enzyme activity in the cells, and to the decrease in glucose concentration by diffusional transport of glucose from the surface to inside the spheroids, as well as the increase in cell density through spheroid formation. These results and our previous studies imply that more malignant cancer cells tend to exhibit glycolytic oscillations with higher frequencies than less malignant cells. Adjacent cells in spheroids oscillated within a 10% difference in frequency, but did not synchronize with each other. This suggests that weak cell‐to‐cell interactions might exist among HeLa cells connected with cadherins in the spheroid microenvironment; however, the interactions were not strong enough to induce synchronization of glycolytic oscillations. Glycolytic oscillations are demonstrated in HeLa cervical cancer cell spheroids. Spheroids are often used to model the in vivo microenvironment of tumours. The properties of spheroids, in particular their metabolic properties, including glycolysis, are reported to be different from those in monolayer cells. The present experimental and mathematical studies revealed the quantitative glycolytic dynamics of HeLa spheroids, which could shed light on the pathophysiological characteristics of cancer.