Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca²⁺/Mn²⁺ transporter regulating both Ca²⁺ and Mn²⁺ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn²⁺ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn²⁺ sensitivity. Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn²⁺ sensitivity.