Microsomal triglyceride transfer protein (MTP) plays a central role in the synthesis of lipoproteins by shuttling lipids between phospholipid membranes to apoB. We have examined the effect of three MTP gene variants, −493G > T, Q95H and H297Q, in 2831 healthy UK middle-aged men. The rare allele frequencies were: 0.25 (95% CI 0.24–0.26) for −493T, 0.054 (95% CI 0.05–0.06) for 95H and 0.32 (95% CI 0.31–0.33) for 297Q. The three variants were in strong allelic association in all pairwise combinations (p < 0.001). None of the variant sites were associated with significant differences in cholesterol, triglyceride, apoB or apoAI levels. When stratified by tertiles of triglycerides for the H297Q variant alone there was a significant effect on apoB levels in men in the top tertile (p = 0.01). Considering the −493G > T and H297Q genotype in combination on baseline levels, individuals with three or four rare alleles had 6.6% higher mean apoB levels compared to the rest (p = 0.007). Therefore, homozygosity for 297Q at higher triglyceride (Tg) levels, or in combination with −493G > T, is associated with a raising effect on apoB levels, suggesting the importance of modest differences in MTP activity in determining hepatic secretion of lipoproteins in healthy men.