Purpose : The purpose of this study was to gain insight into the delivery and immunogenicity of N-trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied transcutaneously with microneedles. ; Methods : Mice were vaccinated with DT-loaded TMC nanoparticles, a solution of TMC and DT (TMC/DT) or DT alone. The formulations were applied onto the skin before or after microneedle treatment with two different 300-µm-long microneedle arrays and also injected intradermally (ID). As a positive control, alum-adjuvanted DT (DT-alum) was injected subcutaneously (SC). Ex vivo confocal microscopy studies were performed with rhodamine-labelled TMC. ; Results : Independent of the microneedle array used and the sequence of microneedle treatment and vaccine application, transcutaneous immunisation with the TMC/DT mixture elicited 8-fold higher IgG titres compared to the TMC nanoparticles or DT solution. The toxin-neutralising antibody titres from this group were similar to those elicited by SC DT-alum. After ID immunisation, both TMC-containing formulations induced enhanced titres compared to a DT solution. Confocal microscopy studies revealed that transport of the TMC nanoparticles across the microneedle conduits was limited compared to a TMC solution. ; Conclusions : In conclusion, TMC has an adjuvant function in transcutaneous immunisation with microneedles, but only if applied in a solution.