Metabolic profiling of the effects of a marginal B‐6 deficiency can provide insight into the sensitivity of 1C metabolism to B‐6 status, and the level of stress placed on different pathways due to this deficiency. It also can add to our knowledge regarding vitamin B‐6 requirement for the optimal performance of the 1C cycle and may generate new hypotheses to be tested. This study aimed to assess the impact of marginal vitamin B‐6 deficiency (plasma PLP 20 –30 nM) on the plasma profile of one‐carbon metabolites in humans. Twenty‐two healthy men and women were placed on a 28‐d controlled dietary vitamin B‐6 restriction. Fasting plasma samples were collected at baseline and at day 28. Targeted analysis of known substrates and products of 1C metabolism and transsulfuration were performed by LC/MS‐MS. A one‐step extraction allowed simultaneous analysis of the concentrations of fifteen metabolites in a small volume of plasma. Results show a significant increase in plasma cystathionine (p<0.01) at marginal B‐6 deficiency, consistent with previous findings from our group. Plasma concentrations of serine and glycine also were significantly higher (p<0.05) at low B‐6 status. Postprandial plasma samples were collected as well and will allow evaluation of substrate availability in the fed versus fasted state on the 1C pathway in a marginal B‐6 deficiency.Supported by NIH grant DK072398 and NIH National Center for Research Resources (NCRR) CTSA grant 1UL1RR029890.