Voltage-gated Na(+) channel (Na(v)) 1.8 contributes to the majority of the Na(+) current that underlies the depolarizing phase of action potentials. Na(v)1.8 is mainly distributed intracellularly and its current amplitude can be enhanced by the beta3 subunit. However, little is known about the mechanisms underlying its intracellular retention and the effects mediated by the beta3 subunit. Here, we show that the beta3 subunit promotes surface expression of Na(v)1.8 by masking its endoplasmic reticulum (ER)-retention/retrieval signal. The RRR motif in the first intracellular loop of Na(v)1.8 is responsible for retaining Na(v)1.8 in the ER and restricting its surface expression. The beta3 subunit facilitates surface expression of Na(v)1.8. The intracellular C-terminus of the beta3 subunit interacts with the first intracellular loop of Na(v)1.8 and masks the ER-retention/retrieval signal. Mutation of the RRR motif results in a significant increase in surface expression of Na(v)1.8 and abolishes the beta3-subunit-mediated effects. Thus, the beta3 subunit regulates surface expression of Na(v)1.8 by antagonizing its ER-retention/retrieval signal. These results reveal a novel mechanism for the effect of the Na(+) channel beta subunits on the alpha subunits.