Transmembrane segment 6 is implicated in slow inactivation (SI) of voltage-gated sodium channels (Navs). To further study its role and understand differences between SI phenotypes of different Nav isoforms, we analyzed several D2-S6 mutants of the human cardiac hNav1.5, which is relatively resistant to SI. Mutants were examined by transient HEK cell transfection and patch-clamp recording of whole-cell Na⁺ currents. Substitutions with lysine (K) included N927K, V930K, and L931K. We show recovery from short (100 ms) depolarization to 0 mV in N927K and L931K is comparable to wild-type, while recovery in V930K is delayed and biexponential, suggesting rapid entry into a slow-inactivated state. SI protocols confirm enhanced SI phenotype (rapid development, hyperpolarized steady-state, slowed recovery) for V930K, contrasting with the resistant phenotype of wild-type hNav1.5. This enhancement, not found in N927K or L931K, suggests that the effect in V930K is site-specific. Glutamine (Q) substituted at V930 also exhibits an enhanced SI phenotype similar to V930K. Therefore, K or Q substitution eliminates hNav1.5 resistance to SI. Alanine (A) or cysteine (C) substitution at V930 show no enhancement of SI, and in fact, V930A and V930C, and L931K, exhibit a resistance to SI, demonstrating that characteristics of specific amino acids (e.g., size, hydrophobicity) differentially affect SI gating. Thus, V930 in D2-S6 appears to be an important structural determinant of SI gating in hNav1.5. We suggest that conformational change involving D2-S6 is a critical component of SI in Navs, which may be differentially regulated between isoforms by other isoform-specific determinants of SI phenotype.