The positive effect of n-3 poly-unsaturated fatty acids (FA) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 PUFA) during in vitro maturation (IVM) has an effect on oocyte maturation and in vitro embryo development in cattle. We showed that DHA (0, 1, 10 or 100 μM) had no effect on oocyte viability or maturation rate after 22 h IVM. Incubation of oocyte-cumulus complexes (OCC) with 1 μM DHA during IVM significantly increased (p<0.05) oocyte cleavage rate as compared to control (86.1% vs. 78.8%, respectively) and the >4-cell embryo rate at day 2 after parthenogenetic activation (PA) (39.1% vs. 29.7%, respectively). Supplementation with 1 μM DHA during IVM also induced a significant increase in the blastocyst rate at day 7 after in vitro fertilization (IVF) as compared to control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; p = 0.08). On the contrary, 10 μM DHA had no effects, whereas 100 μM DHA significantly decreased the cleavage rate compared to control (69.5% vs.78.8%, respectively) and the >4-cell embryo rate at day 2 after PA (19.5% vs. 29.7%). As was shown by real-time PCR, negative effects of 100 μM DHA were associated with significant increase of progesterone synthesis by OCCs, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CC) of corresponding oocytes. DHA at 1 and 10 μM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1μM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding cumulus cells, contrarily to 100 μM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.