Soil pH and the availability of carbon (C) substrate and nutrients to microorganisms are well recognized to influence C priming. However, the mechanisms underpinning such interplay so far remain elusive. Given that liming acid soils, residue retention and fertilization are common agricultural practices, small changes in SOC content by these practices could have a big impact on the global C budget. This study aimed to gain insight into the impact of initial pH and mineral N availability on the priming effect of two C substrates with contrasting biodegradability. Stable 13C-labelled substrates, glucose and lignocellulose, were applied at the rate of 0.5 mg C g−1 soil with or without NH4NO3 to the same soil matrix with three different initial pH levels; pH 4.1 (strongly acidic), 4.7 (moderately acidic) and 6.6 (slightly acidic). The N treatment was based on a C:N of 10 of the added substrate (0.05 mg N g−1 soil) to ensure N was non-limiting. Interestingly, the priming effect was not linearly related to soil pH; greatest at pH 4.1, followed by pH 6.6 and lowest at pH 4.7. The greater net increase in microbial biomass upon C supply in strongly acidic soils compared to the moderately and slightly acidic soils would have enhanced co-metabolic decomposition of native soil organic C (SOC). The cumulative amount of primed SOC during the 30-day incubation period was greater in glucose- (21 μg C g−1) than lignocellulose-amended soils (13 μg C g−1). Nitrogen application reduced the C priming effect of both C substrates at all pH levels. This reduction was more prominent with lignocellulose and in the moderately acidic soils. The results suggest that maintaining optimal soil pH for nutrient availability and N application that exceeds the microbial N requirements in agricultural fields may minimize SOC loss via the priming effect in the short term.