Isoniazid (INH), used in the standard treatment of tuberculosis, competes against vitamin B6 (VB6) and decreases its plasma levels. The present study aims to analyze changes in plasma VB6 (pyridoxal) levels during tuberculosis treatment because of limited availability of such data. Among 66 patients with active pulmonary tuberculosis, 14 were eligible for this study. All patients consumed a standard hospital diet during the study period. Of the 10 patients aged >65 years, 9 were malnourished and 1 suffered from chronic kidney disease. Plasma VB6 levels were below normal in 12 patients before INH administration, and these levels decreased further at 2 and 4 weeks after INH administration. However, no signs or symptoms of peripheral neuropathy were observed in any patient. Chronic VB6 deficiency may thus induce several adverse effects, except for peripheral neuropathy. Further randomized control studies should be conducted to clarify the need for supplemental VB6 during tuberculosis treatment using INH.