A measurement of D0 meson production by the LHCb experiment in its fixed-target configuration is presented. The production of D0 mesons is studied with a beam of 2.5  TeVTeV protons colliding on a gaseous neon target at rest, corresponding to a nucleon–nucleon centre-of-mass energy of √sNN=68.5  GeV. The sum of the D0 and D‾0 production cross-section in pNe collisions in the centre-of-mass rapidity range y⋆∈[−2.29,0] is found to be \sigma _{D^{0}}^{y^\star \in [-2.29, 0]} = 48.2 \pm 0.3 \pm 4.5 ,\upmu \text {b}/\text {nucleon} where the first uncertainty is statistical and the second is systematic. The D0−D‾0 production asymmetry is also evaluated and suggests a trend towards negative values at large negative y⋆. The considered models do not account precisely for all the features observed in the LHCb data, but theoretical predictions including 1% intrinsic charm and 10% recombination contributions better describe the data than the other models considered.