Abnormalities in tricarboxylic acid (TCA) cycle function were related to a variety of pathological processes. Fumarate hydratase (FH) is a required enzyme in the TCA cycle. To explore the general influence of FH knockout, we isolated FH+/– rat and normal rat lung fibroblasts and cultured these cells in vitro. The isolated fibroblasts with the current method were rather homogeneous and were confirmed spindle in morphology, positive for vimentin and negative for α-SMA (α-smooth muscle actin). Sequencing of the PCR (polymerase chain reaction) products flanking the FH gene mutation verified the FH+/– status, and the FH gene and protein expression were confirmed to be reduced in the FH+/– cells. No sign of ageing for the FH+/– cells after 61 passages was observed, but the controls died out at this stage. Flow cytometry revealed increased S-phase and decreased G1/G0 proportions with significantly less early apoptosis in FH+/– cells compared to that in control cells. At the same time, increased glucose consumption, intracellular fumarate production and extracellular lactate secretion were verified in the FH+/– cells. Correspondingly, FH+/– cells showed a lower basal oxygen consumption rate (OCR) but a higher level of reactive oxygen species (ROS) production. Single cell cloning and cell line establishment were successfully performed with the FH+/– cells at the 84th passage. All the above results indicate an important role for FH+/– in the longevity or immortality of the FH+/– cells, in which increased p53 and TERT (telomerase reverse transcriptase) protein expression, decreased p21 and p16 protein expression and negative SA-β-Gal (senescence-associated beta-galactosidase) were verified along with metabolic reprogramming.