Formation of NO initiated by heterogeneous fixation of N2 during pyrolysis is investigated experimentally and theoretically. The experiments were conducted with beech wood as well as with the pure biomass components cellulose, xylan, and lignin. The NO formation during char oxidation was recorded as function of pyrolysis atmosphere (N2 or Ar), pyrolysis temperature (700–1050 °C), and oxidizing atmosphere (O2 in N2 or Ar). The results confirm earlier reports that biomass char may be enriched in N during pyrolysis at 900 °C and above. The N-uptake involves re-capture of N-volatiles as well as uptake of N2. During char oxidation, the captured N is partly oxidized to NO, resulting in increased NO formation. The NO yield from oxidation of beech wood char made in N2 increases with pyrolysis temperature, and is about a factor of two higher at 1050 °C than the corresponding yield from chars made in Ar. The experiments with pure materials show that the lignin char has the strongest ability to form NO from uptake of N2, while xylan char forms only small amounts of NO from N2. Density Functional Theory (DFT) calculations on model chars have revealed a number of chemisorption sites for N2, many of which are weakly bound and therefore expected to have a short half-life at the higher pyrolysis temperatures. However, the chemisorption of N2 across a single ring of the armchair surface was found to have an activation energy of 344 ± 30 kJ mol−1 and form a stable, exothermic product with cyano groups. This demonstrates that at least one channel exists for the high-temperature incorporation of N2 into a char which could give rise to the observed increase in NO release in subsequent char oxidation.