Background <p>This study tested the hypothesis that mitofusin-2 overexpression (Mfn2^Ove) in adipose-derived mesenchymal stem cells (ADMSC^Ove−Mfn2) protects the testis against testicular torsion-induced ischemia‒reperfusion (TTIR) injury via AMP-activated protein kinase (AMPK)-mediated mitochondrial dynamic homeostasis.</p> Methods and results <p>The in vitro results revealed that Mfn2^Ove significantly increased cell viability/mitochondrial content/fusion protein/ATP levels (all <i>P</i> &lt; 0.001). Mfn2^Ove in ADMSCs and GC-1 cells upregulated the expression of mitochondrial fusion proteins/content/OXPHOS complexes/p-AMPK and suppressed fission proteins/autophagy/oxidative stress, which were reversed by siRNA-Mfn2 (all <i>P</i> &lt; 0.001). Compared with the control, Mfn2^Ove significantly increased mitochondrial length in ADMSCs and GC-1 cells (<i>P</i> &lt; 0.0001). IR activated p-AMPK and upregulated fission proteins, whereas the AMPK inhibitor did not reverse the ability of Mfn2^Ove to upregulate p-AMPK/Mfn2/OPA1/OXPHOS/mitochondrial length in ADMSCs and GC-1 cells (<i>P</i> &lt; 0.0001). By day 28 after TTIR induction, testicular protein levels of oxidative stress/fibrosis/apoptosis/autophagy/DNA-damage/fission were significantly increased in group 2 (TTIR) compared with those of group 1 (sham-control), whereas the protein levels of mitochondrial fusion/OXPHOS exhibited an opposite pattern of oxidative stress between groups 1 and 2 that were significantly reversed by AMDCs (group 3) and further reversed by ADMSC^OVe−Mfn2 (group 4), which was reversed by siRNA-Mfn2 in ADMSCs (group 5) (all <i>P</i> &lt; 0.00001). The p-AMPK protein level significantly increased from groups 1 to 4 but was reversed in group 5 (all <i>P</i> &lt; 0.00001). The cellular levels of androgen receptor/Sertoli cells/alpha-microtubule/vimentin/testis injury score showed identical patterns to those of OXPHOS complexes among the groups (all <i>P</i> &lt; 0.00001).</p> Conclusion <p>The activation of the AMPK–Mfn2 axis protected the testis against TTIR injury.</p> Graphical Abstract <p></p>