Connexin 33 (Cx33) is a testis‐specific gap junction protein. We previously reported that Cx33 exerts dominant‐negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild‐type Sertoli cells transfected with Cx33‐DsRed2 and Cx43‐green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi‐channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy‐fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens‐1 (ZO‐1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO‐1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO‐1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO‐1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant‐negative effect on gap junction plaque.