(1) Background: Oxidative energy metabolism is presumed to rely on the optimal iron supply. Primary human cardiac myocytes (HCM) exposed to different iron availability conditions during mechanical stretch are anticipated to demonstrate expression changes of genes involved in aerobic and anaerobic metabolic pathways. (2) Methods: HCM were cultured for 48 h either in static conditions and upon mechanical stretch at the optimal versus reduced versus increased iron concentrations. We analyzed the expression of pyruvate kinase (PKM2), lactate dehydrogenase A (LDHA), and mitochondrial complexes I–V at the mRNA and protein levels. The concentration of l
-lactate was assessed by means of lactate oxidase method-based kit. (3) Results: Reduced iron concentrations during mechanical work caused a decreased expression of complexes I–V (all p
< 0.05). The expression of PKM2 and LDHA, as well as the medium concentration of l
-lactate, was increased in these conditions (both p
< 0.05). HCM exposed to the increased iron concentration during mechanical effort demonstrated a decreased expression of mitochondrial complexes (all p
< 0.01); however, a decrement was smaller than in case of iron chelation (p
< 0.05). The iron-enriched medium caused a decrease in expression of LDHA and did not influence the concentration of l
-lactate. (4) Conclusions: During mechanical effort, the reduced iron availability enhances anaerobic glycolysis and extracellular lactate production, whilst decreasing mitochondrial aerobic pathway in HCM. Iron enrichment during mechanical effort may be protective in the context of intracellular protein machinery of non-oxidative metabolism with no effect on the extracellular lactate concentration.