Circadian rhythms affect the total cholesterol levels in humans and animals, although their effect on cholesterol synthesis remain poorly understood. Here, we show for the first time that intermediates of the post-squalene portion of cholesterol synthesis also follow a circadian rhythm in the mouse liver. We used Crem-knock-out mice to investigate the effects of cAMP response element modulator (CREM) isoforms on cholesterol synthesis over time, as compared to wild-type mice. Multiple linear regression and cosinor statistical analysis were carried out on data obtained from 166 liver samples of mice, and the 24-h profiles were modelled across genotype, gender and zeitgeber time for lanosterol, 24,25-dihydrolanosterol, testis meiosis-activating sterol, and 7-dehydrocholesterol, along with cholesterol. The levels of these sterols were higher in female mice compared to males, although the genotype/gender factors showed no effects on the circadian oscillation of these sterols, except for 24,25-dihydrolanosterol. This study also highlights the importance of the statistical methods, where time, genotype and gender are the studied variables.