Monitoring genotypic and phenotypic stability is crucial during the development of recombinant Chinese hamster ovary (CHO) cell lines. Although genotypic instability is well‐studied, there are few reports on phenotypic instability. Here, a case study of two clonal cell lines derived from Pfizer's site‐specific integration expression platform that expresses the same monoclonal antibody is described. It is shown that both cell lines (herein referred to as “Cell Line A” and “Cell Line B”) are genotypically stable up to 130 generations. However, when both cell lines are run side‐by‐side in a fed‐batch production assay, productivity from Cell Line A later generation cells is much lower when compared to earlier generation cells. Phenotypically, later generation Cell Line A cells display increased lactate production, decreased productivity, and decreased cell viability. Metabolic analysis reveals that Cell Line A exhibits increased glycolysis activity and capacity at higher generational age. Whole transcriptomic sequencing shows significant upregulation of the hypoxia‐inducible factor 1‐alpha (HIF‐1α) signaling pathway and associated downstream targets. Furthermore, Western blot analysis confirms elevated HIF‐1α protein in Cell Line A cells at later generation. These results suggest a novel role for HIF‐1α in the age‐associated metabolic changes that result in the phenotypic instability of a recombinant CHO cell line.