The purpose of this study was to investigate potential mechanisms involved in fat deposition promoted by dietary lysine deficiency, particularly intramuscular fat (IMF), and differential responses between fatty and lean pigs. Carcass traits and lipogenic enzyme activities and gene expression levels in muscles and adipose tissue were investigated in Iberian (fatty) and Landrace × Large White (LDW) pigs under identical feeding level (based on body weight (BW)) and management conditions. Twenty-eight barrows of 10 kg initial BW, 14 per breed, were fed two isoproteic (200 g CP /kg DM) and isocaloric (14.7 MJ metabolizable energy/kg DM) diets with identical composition except for the lysine content (1.09% for diet adequate in lysine and 0.52% for diet deficient in lysine). At a BW of 25 kg, pigs were slaughtered. Compared with pigs fed the lysine-adequate diet, in both genotypes lysine-deficient diet led to lower carcass protein concentration, lower relative proportions of leaner components (loin, ham and shoulder; P < 0.01), and higher carcass fatty components and carcass lipid concentration (P < 0.001). Irrespective of diet, the activity and gene expression of lipogenic enzymes (fatty acid synthase (FAS), malic enzyme (ME) and glucose-6-phosphate dehydrogenase (G6PDH)) were greater in Iberian than in LDW pigs, particularly in adipose tissue where transcriptional regulators involved in the control of adipogenesis and lipogenesis were also upregulated in Iberian animals. In backfat tissue, there was a small decrease induced by or no effects of lysine-deficient diet on the activity and gene expression of lipogenic enzymes, nor in gene expression levels of upstream regulators of lipogenesis and adipogenesis. In longissimus muscle, the activity of FAS, G6PDH and ME increased with lysine deficiency in both genotypes (P < 0.01) and an upregulation of gene expression of lipogenic enzymes was specifically observed in Iberian pigs (P < 0.05 to P < 0.001). In biceps femoris muscle of lysine-deficient pigs, the activity of FAS and ME enzymes increased, ME1 gene was upregulated (added to FASN gene in the case of Iberian pigs; P < 0.01 to P < 0.001) and PPARA gene was downregulated (P < 0.05). The results show that in both fatty and lean pigs, the effect of lysine deficiency on lipid metabolism was tissue-specific, with an activation of lipogenesis in longissimus and biceps femoris muscle but no apparent stimulation in backfat adipose tissue. Suitable feeding protocols including lysine-deficient diets should be designed for each pig type in order to increase intramuscular lipids without penalizing the growth of lean carcass components.