Exploring the metastable magnetic nanostructures of Mn substituted α″-Fe16N2 with large saturation magnetization μ0MS, high Curie temperature TC and giant magnetic anisotropy are of technological merit as promising candidates for non-rare-earth based permanent magnets. Here, we present in-depth analysis for the structural and magnetic properties of Fe16 − xMnxN2 using first-principles calculations. We predict a large magnetocrystalline anisotropy energy (MAE) constant of K1 = 2.02 MJ/m3 for the Fe14Mn2N2 alloy, which is more than twice that of pristine Fe16N2. The underlying mechanism associated with boosting K1 is attributed to the local distortion of orbitals induced by Mn substitution. The MAE is also carefully analyzed in terms of reciprocal space analysis by employing the magnetic force theorem, revealing the regions in the Brillouin zone that are prominent for giving rise to MAE.