Incorporation of molecular adjuvants into DNA vaccines is often used to improve the induction of immune responses, but few approaches aim to specifically activate B cells for an enhanced humoral response only. Hemokinin-1 (HK-1) is a factor that activates B cells for proliferation, survival, differentiation into plasma cells, and Ab production. Therefore, we investigated if it may be used as a molecular adjuvant for DNA vaccines to elicit strong humoral and memory responses. The HK-1 coding sequence was sub-cloned as single or triple copies in-frame downstream of S2 HBsAg in the proVAX/S2 construct. Compared to mice immunized with proVAX/S2 or proVAX/S2-HK-1, proVAX/S2-3HK-1 induced a higher level of IgG production, a higher percentage of differentiated antibody-secreting plasma cells, and a higher level of T-cell proliferation. Furthermore, a higher proportion of B cells had the B220(+)CD27(+) phenotype in these groups, and specific antigen re-challenge induced a higher level of total IgG production 60 d after the last immunization, suggesting that the use of HK-1 as an adjuvant promoted immunological memory. Taken together, these results suggest that using HK-1 as an adjuvant molecule could enhance the immunogenicity of HBsAg DNA vaccines, and result in stronger humoral and memory responses. Therefore, HK-1 may lead to the development of a novel humoral-biased molecular adjuvant for an HBsAg DNA vaccine against hepatitis B infection.