The aim of the present study was to evaluate the protective effect of L-glutamine (L-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, L-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk - EY - 20%, and glycerol 3%). No significant improvement (P>0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility - CASA system - and flow cytometric analysis of plasma and acrosomal membrane integrity -SYBR14/PI/PE-PNA- and plasma membrane stability -M540/YoPro1-). In Experiment 2, L-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P<0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM L-Gln compared to control (3% glycerol). In Experiment 3, L-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of L-Gln and the absence of EY. In conclusion, L-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.