Fluorescent dyes possessing a variety of arylacetylenes at the 9-position of a xanthene skeleton were synthesized and their optical properties were investigated. The π system effectively expanded over the xanthene skeleton and the aryl group through the triple bond. Starting from the emission wavelength (λem) of 9-methyl xanthene 20 in basic DMSO solution at 536 nm, the emission wavelengths gradually shifted to the red region for methylacetylene 17 (λem = 600 nm), phenylacetylene 5 (λem = 636 nm), and p-CF3-phenylacetylene 11 (λem = 660 nm). On the basis of these data, we estimated the substituent effects for the red shift on the emission wavelength and rationally explained the results by DFT calculations. Furthermore, potential applicability of these fluorescent dyes to cell staining was exemplified.