Camelina oil (CO) is a potential replacement for fish oil (FO) in aquaculture feeds. CO is high in α-linolenic acid (18:3n-3 or ALA) (35%), with an omega-3/omega-6 (n-3/n-6) ratio near 2. In order to test the effect of CO on the overall performance of tilapia (Oreochromis niloticus var. GIFT), feed utilization, lipid composition and capacity to synthesize the long-chain fatty acids eicosapentaenoic acid (20:5n-3 or EPA) and docosahexaenoic acid (22:6n-3 or DHA) from ALA, were tested in an 8-week feeding trial with juvenile tilapia. The average fish weight at the start was 28 ± 6 g and they were grown in a biofloc system. Four dietary treatments were formulated, two containing either fish oil (TFO) or camelina oil (TCO), and two more where FO was replaced by CO at low (Low-CO) and mid (Mid-CO) levels. A commercial diet (COM) was used as a reference diet. Compound-specific stable isotope analysis (CSIA) and stable isotope mixing models with R software (SIAR) were used to calculate the contribution of ALA to EPA and DHA synthesis. At the end of the experiment, replacing FO by CO had no effect on growth (139 ± 22 g fish−1) or total lipid in the muscle (2.2–2.9 g). However, the tilapia fed TCO had significantly more phospholipid in muscle compared to tilapia fed TFO. Also, a higher content of linoleic acid (18:2n-6 or LOA) and ALA was revealed. ALA content in muscle followed the ALA content in diets; by contrast, EPA and DHA decreased significantly as the level of dietary CO increased. Despite the variation in fatty acids, n-3 PUFA and the n-3/n-6 ratio in muscle tissue did not show differences among experimental diets. CSIA revealed that the δ13C isotopic signature of DHA in tilapia muscle after feeding TCO and biofloc was slightly but significantly enriched in (Budge et al., 2008)C. However, CO feeding resulted in a significantly depleted isotopic signal for docosapentaenoic acid (22:5n-3 or DPA) compared to FO. SIAR indicated that 28% of DHA, 36% of EPA, and 40% of DPA was synthesized from camelina oil ALA. •The total replacement of fish oil by camelina oil in the diet for tilapia not decreased the growth performance.•Camelina oil not increased EPA + DHA in fillet as fish oil did but produced an adequate n-3/n-6 ratio for human diet.•The CSIA showed that 28–40% of n-3 LC-PUFA in tilapia muscle could be synthesized from camelina oil ALA.