Current bottom–up process models suggest that montane tropical ecosystems are weak atmospheric sources of N2O, although recent empirical studies from the southern Peruvian Andes have challenged this idea. Here we report N2O flux from combined field and laboratory experiments that investigated the process-based controls on N2O flux from montane ecosystems across a large-elevation gradient (600–3700 m a.s.l.) in the southern Peruvian Andes. Nitrous oxide flux and environmental variables were quantified in four major habitats (premontane forest, lower montane forest, upper montane forest and montane grassland) at monthly intervals over a 30-month period from January 2011 to June 2013. The role of soil moisture content in regulating N2O flux was investigated through a manipulative, laboratory-based 15N-tracer experiment. The role of substrate availability (labile organic matter, NO3−) in regulating N2O flux was examined through a field-based litter-fall manipulation experiment and a laboratory-based 15N–NO3− addition study, respectively. Ecosystems in this region were net atmospheric sources of N2O, with an unweighted mean flux of 0.27 ± 0.07 mg N–N2O m−2 d−1. Weighted extrapolations, which accounted for differences in land surface area among habitats and variations in flux between seasons, predicted a mean annual flux of 1.27 ± 0.33 kg N2O–N ha−1 yr−1. Nitrous oxide flux was greatest from premontane forest, with an unweighted mean flux of 0.75 ± 0.18 mg N–N2O m−2 d−1, translating to a weighted annual flux of 0.66 ± 0.16 kg N2O–N ha−1 yr−1. In contrast, N2O flux was significantly lower in other habitats. The unweighted mean fluxes for lower montane forest, montane grasslands, and upper montane forest were 0.46 ± 0.24 mg N–N2O m−2 d−1, 0.07 ± 0.08 mg N–N2O m−2 d−1, and 0.04 ± 0.07 mg N–N2O m−2 d−1, respectively. This corresponds to weighted annual fluxes of 0.52 ± 0.27 kg N2O–N ha−1 yr−1, 0.05 ± 0.06 kg N2O–N ha−1 yr−1, and 0.04 ± 0.07 kg N2O–N ha−1 yr−1, respectively. Nitrous oxide flux showed weak seasonal variation across the region; only lower montane forest showed significantly higher N2O flux during the dry season compared to wet season. Manipulation of soil moisture content in the laboratory indicated that N2O flux was significantly influenced by changes in water-filled pore space (WFPS). The relationship between N2O flux and WFPS was complex and non-linear, diverging from theoretical predictions of how WFPS relates to N2O flux. Nitrification made a negligible contribution to N2O flux, irrespective of soil moisture content, indicating that nitrate reduction was the dominant source of N2O. Analysis of the pooled data indicated that N2O flux was greatest at 90 and 50 % WFPS, and lowest at 70 and 30 % WFPS. This trend in N2O flux suggests a complex relationship between WFPS and nitrate-reducing processes (i.e. denitrification, dissimilatory nitrate reduction to ammonium). Changes in labile organic matter inputs, through the manipulation of leaf litter-fall, did not alter N2O flux. Comprehensive analysis of field and laboratory data demonstrated that variations in NO3− availability strongly constrained N2O flux. Habitat – a proxy for NO3− availability under field conditions – was the best predictor for N2O flux, with N-rich habitats (premontane forest, lower montane forest) showing significantly higher N2O flux than N-poor habitats (upper montane forest, montane grassland). Yet, N2O flux did not respond to short-term changes in NO3− concentration.