African Swine Fever (ASF) is a highly contagious disease caused by a double-stranded DNA virus (ASFV). Despite significant advances made over the last decade, issues such as residual virulence and absence of differentiating infected from vaccinated animals (DIVA) capacity remain an obstacle in the development of live attenuated vaccines (LAVs) against ASFV. It is, therefore, necessary to identify novel strategies to improve vaccine safety, by rational mutagenesis of virulence associated genes and generation of DIVA markers. ASFV encodes a HU (histone-like protein from E. coli strain U93) homolog protein, pA104R, which is involved in viral genome assembly and host immune recognition. A phylogenetic analysis revealed that pA104R is highly conserved among ASFV isolates, suggesting that it can be a good target for vaccine design. Thus, we selectively mutated the β-strand DNA binding region (BDR) of pA104R to attenuate its enzymatic activity, and identified and mutated several B-cell epitopes present in pA104R to generate a negative marker. Residues K64, K66, and R69 in the BDR were identified as relevant for pA104R activity, with double mutation of the first two showing additive attenuation. pA104R-reactive IgM and IgG epitopes were also identified in the bottom of the BDR, with selective mutagenesis drastically reducing antibody recognition and, when combined with mutations in the arm of the BDR, leading to a further reduction of DNA-binding activity. Interestingly, the immunodominant pA104R-reactive IgG epitope was mainly recognized by IgG1 suggesting that pA104R induces a dominant Th2 response. In sum, the rational mutagenesis can reduce pA104R-DNA binding activity and immune reactivity, providing a rationale for the development of an ASFV pA104R-based DIVA vaccine.