Synthetic 21‐bp‐long short interfering RNAs (siRNA) can stimulate sequence‐specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain‐related cation‐channel P2X₃ expressed in CHO‐K1 and a rat brain tumour‐derived cell line, 33B. Downregulation of the P2X₃ receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence‐specific inhibition of the P2X₃ mRNA, independent of the type of 2 nt 3′‐overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi‐mediated specific inhibition of the P2X₃ receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X₃ mRNA. The two reagents resulted in more efficient downregulation of P2X₃ mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi‐mediated treatment for inhibiting expression of a medically relevant pain‐related gene.