The CHRM3 gene is a member of the muscarinic acetylcholine receptor family that plays important roles in the regulation of fundamental physiological functions. The evolutionary mechanism of exon-acquisition and alternative splicing of the CHRM3 gene in relation to transposable elements (TEs) were analyzed using experimental approaches and in silico analysis. Five different transcript variants (T1, T2, T3, T3-1, and T4) derived from three distinct promoter regions (T1: L1HS, T2, T4: original, T3, T3-1: THE1C) were identified. A placenta (T1) and testis (T3 and T3-1)-dominated expression pattern appeared to be controlled by different TEs (L1HS and THE1C) that were integrated into the common ancestor genome during primate evolution. Remarkably, the T1 transcript was formed by the integration event of the human specific L1HS element. Among the 12 different brain regions, the brain stem, olfactory region, and cerebellum showed decreased expression patterns. Evolutionary analysis of splicing sites and alternative splicing suggested that the exon-acquisition event was determined by a selection and conservation mechanism. Furthermore, continuous integration events of transposable elements could produce lineage specific alternative transcripts by providing novel promoters and splicing sites. Taken together, exon-acquisition and alternative splicing events of CHRM3 genes were shown to have occurred through the continuous integration of transposable elements following conservation.