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The aims of this study were to examine the effects of pyridoxine administration on the activities of cardiac antioxidant stress enzymes superoxide dismutase (SOD) and catalase (CAT) and enzyme indicators of cardiometabolic status, lactate and malate dehydrogenase (LDH, MDH), as well as LDH and MDH isoforms’ distribution in the cardiac tissue of healthy and diabetic Wistar male rats. Experimental animals were divided into five groups: C1—control (0.9% sodium chloride—NaCl—1 mL/kg, intraperitoneally (i.p.), 1 day); C2—second control (0.9% NaCl 1 mL/kg, i.p., 28 days); DM—diabetes mellitus (streptozotocin 100 mg/kg in 0.9% NaCl, i.p., 1 day); P—pyridoxine (7 mg/kg, i.p., 28 days); and DM + P—diabetes mellitus and pyridoxine (streptozotocin 100 mg/kg, i.p., 1 day and pyridoxine 7 mg/kg, i.p., 28 days). Pyridoxine treatment reduced CAT and MDH activity in diabetic rats. In diabetic rats, the administration of pyridoxine increased LDH1 and decreased LDH4 isoform activities, as well as decreased peroxisomal MDH and increased mitochondrial MDH activities. Our findings highlight the positive effects of pyridoxine administration on the complex interplay between oxidative stress, antioxidant enzymes, and metabolic changes in diabetic cardiomyopathy.