Climate change currently manifests in upward and northward shifting treelines, which encompasses changes to the carbon (C) and nitrogen (N) composition of organic inputs to soils. Whether these changed inputs will increase or decrease microbial mineralisation of native soil organic matter remains unknown, making it difficult to estimate how treeline shifts will affect the C balance. Aiming to improve mechanistic understanding of C cycling in regions experiencing treeline shifts, we quantified priming effects in soils of high altitudes (Peruvian Andes) and high latitudes (subarctic Sweden), differentiating landcover types (boreal forest, tropical forest, tundra heath, Puna grassland) and soil horizons (organic, mineral). In a controlled laboratory incubation, soils were amended with substrates of different C:N, composed of an organic C source at a constant ratio of 30% substrate-C to microbial biomass C, combined with different levels of a nutrient solution neutral in pH. Substrate additions elicited both positive and negative priming effects in both ecosystems, independent from substrate C:N. Positive priming prevailed above the treeline in high altitudes and in mineral soils in high latitudes, where consequently climate change-induced treeline shifts and deeper rooting plants may enhance SOM-mineralisation and soil C emissions. However, such C loss may be compensated by negative priming, which dominated in the other soil types and was of larger magnitude than positive priming. In line with other studies, these results indicate a consistent mechanism linking decreased SOM-mineralisation (negative priming) to increased microbial substrate utilisation, suggesting preferential substrate use as a potential tool to support soil C storage.