Circadian rhythms are self-sustained physiological changes that drive rhythmicity within the 24-hours cycles. Posttranslational modifications (PMTs), such as protein phosphorylation, acetylation, sumoylation, and ubiquitination, are biochemical processes that modify protein structure and functions, ensuring circadian rhythm precision. For example, phosphorylation is considered the most important hallmark of rhythmicity from cyanobacteria to mammals. Cyclin-dependent kinase 5 (CDK5) has been shown to regulate the mammalian SCN's circadian clock via phosphorylation of PER2. Here, we show that CDK5 influences the clock machinery assembling, using immortalized mouse embryonic fibroblast as an in vitro model for studying the peripheral clock. In fact, the circadian period at the cellular level is lengthened. Furthermore, the clock-controlled gene's expression amplitude is dampened in Cdk5 ko cell lines, while the phase is delayed about 4 hours. Taken together, we show in vitro that CDK5 is critically involved in regulating the peripheral clocks, influencing their temporal and spatial dynamics.