Will warming lead to an increased use of older soil organic carbon (SOC) by microbial communities, thereby inducing C losses from C-rich alpine soils? We studied soil microbial community composition, activity and substrate use after three and four years of soil warming (+4°C, 2007-2010) at the alpine treeline in Switzerland. The warming experiment was nested in a free air CO2 enrichment experiment using depleted (13) CO2 (δ(13) C = -30‰, 2001-2009). We traced this depleted (13) C label in phospholipid fatty acids (PLFA) of the organic layer (0-5 cm soil depth) and in C mineralized from root-free soils to distinguish substrate ages used by soil microorganisms: fixed before 2001 ("old"), from 2001 to 2009 ("new") or in 2010 ("recent"). Warming induced a sustained stimulation of soil respiration (+38%) without decline in mineralizable SOC. PLFA concentrations did not reveal changes in microbial community composition due to soil warming, but soil microbial metabolic activity was stimulated (+66%). Warming decreased the amount of new and recent C in the fungal biomarker 18:2ω6,9 and the amount of new C mineralized from root-free soils, implying a shift in microbial substrate use towards a greater use of old SOC. This shift in substrate use could indicate an imbalance between C inputs and outputs, which could eventually decrease SOC storage in this alpine ecosystem. This article is protected by copyright. All rights reserved.