The use of plant growth regulators (PGRs) is widespread in commercial table grape vineyards. The synthetic cytokinin CPPU is a PGR that is extensively used to obtain higher quality grapes. However, the effect of CPPU on berry firmness is not clear. The current study investigated the effects of pre-anthesis applications (BBCH15 and BBCH55 stages) of CPPU on ‘Thompson Seedless’ berry firmness at harvest through a combination of cytological, morphological, and biochemical analyses. Ovaries in CPPU-treated plants presented morphological changes related to cell division and cell wall modification at the anthesis stage (BBCH65). Moreover, immunofluorescence analysis with monoclonal antibodies 2F4 and LM15 against pectin and xyloglucan demonstrated that CPPU treatment resulted in cell wall modifications at anthesis. These early changes have major repercussions regarding the hemicellulose and pectin cell wall composition of mature fruits, and are associated with increased calcium content and a higher berry firmness at harvest.