The neutral lipid of desilked eri silkworm pupae (Samia cynthia ricini) grown on two different host plants, castor (Ricinus communis Linn.) and tapioca (Manihot utilizsima Phol.) leaves, was extracted with hexane. The oil content in pupae was estimated to be in the range of 18-20% (dry basis). The pupal oil was found to be enriched with alpha-linolenic acid (ALA) with palmitic acid as the second major fatty acid. The level of ALA in the oil of silkworm pupae was found to be significantly higher (P < 0.001) when grown on tapioca (58.3%) as compared to those grown on castor (42.9%). Other chemical parameters such as percent free fatty acid, peroxide value, phosphorus content, percent unsaponifiable matter, and composition of sterols were also determined in both of the oils and compared. Reversed-phase high-performance liquid chromatography analysis of triacylglycerol molecular species showed that the pupal oil is rich in molecular species with equivalent carbon numbers (ECN) C36, C40, C42, C44, and C48. There was a significantly higher level (P < 0.001) of trilinolenin (C36) in the oil of tapioca-based silkworm as compared to castor-based silkworm pupae. Regiospecific analysis of the oil showed a higher level of ALA at the sn-2 position of silkworm pupae grown on tapioca (60.2%) as compared to those grown on castor (47.3%) oil. Thus, the presence of a large amount of ALA and their predominance at the sn-2 position make the eri pupal oil highly nutritious, provided that the oxidative stability is ensured.