Background The molecular mechanism of class-switch recombination (CSR) in human subjects has not been fully elucidated. The CSR-induced mutations occurring in the switch region of the IgM gene (Smu-SHMs) in in vitro CSR-activated and in vivo switched B cells have been analyzed in mice but not in human subjects. Objective We sought to better characterize the molecular mechanism of CSR in human subjects. Methods Smu-SHMs were analyzed in vitro and in vivo by using healthy control subjects and patients with molecularly defined CSR defects. Results We found that Smu-SHMs can be induced in vitro by means of CSR activation in human subjects. We also found large amounts of Smu-SHMs in in vivo class-switched memory B cells, smaller (although significant) amounts in unswitched memory B cells, and very low amounts in naive B cells. In class-switched memory B cells a high frequency of Smu-SHMs was found throughout the Smu. In unswitched memory B cells, the Smu-SHM frequency was significantly decreased in the 5′ part of the Smu. The difference between switched and unswitched B cells suggests that the extension of somatic hypermutation (SHM) to the 5′ upstream region of the Smu might be associated with the effective induction of CSR. The analysis of the pattern of mutations within and outside the WRCY/RGYW (W, A/T; R, A/G; and Y, C/T) motifs, as well as the Smu-SHMs, in CD27+ B cells from CD40 ligand (CD40L)–, activation-induced cytidine deaminase (AID)–, and uracil-DNA glycosylase (UNG)–deficient patients revealed the dependence of Smu-SHM on CD40L, AID, UNG, and the mismatch repair system in human subjects. Conclusion CD40L-, AID-, UNG-, and mismatch repair system–dependent Smu-SHMs and extension to the 5′ region of Smu are necessary to accomplish effective CSR in human subjects.