Background: Malania oleifera Chun et Lee (Olacaceae), an evergreen broad-leaved woody tree native to southwest China, is an important oilseed tree. Its seed oil has a high level of nervonic acid (cis-tetracos-15-enoic acid, over 60%), which is essential for human health. M. oleifera seed oil is a promising source of nervonic acid, but little is known about the physiological and molecular mechanisms underlying its biosynthesis. Results: In this study, we recorded oil accumulation at four stages of seed development. Using a high-throughput RNA-sequencing technique, we obtained 55,843 unigenes, of which 29,176 unigenes were functionally annotated. By comparison, 22,833 unigenes had a two-fold or greater expression at the fast oil accumulation stage than at the initial stage. Of these, 198 unigenes were identified as being functionally involved in diverse lipid metabolism processes (including de novo fatty acid synthesis, carbon chain elongation and modification, and triacylglycerol assembly). Key genes (encoding KCS, KCR, HCD and ECR), putatively responsible for nervonic acid biosynthesis, were isolated and their expression profiles during seed development were confirmed by quantitative real-time PCR analysis. Also, we isolated regulatory factors (such as WRI1, ABI3 and FUS3) that are putatively involved in the regulation of oil biosynthesis and seed development. Conclusion: Our results provide novel data on the physiological and molecular mechanisms of nervonic acid biosynthesis and oil accumulation in M. oleifera seeds, and will also serve as a starting point for biotechnological genetic engineering for the production of nervonic acid resources.