Background: Gracilaria lemaneiformis is an edible red marine macroalga that contains various active components including phycoerythrin, polysaccharides, and phenolics. In our previous work, crude ethanolic extracts of G. lemaneiformis exhibited potential antioxidative and anti-photoaging activities. Therefore, in this study, we aimed to further investigate the antioxidative and anti-photoaging activities of different fractions of G. lemaneiformis (n-hexane, ethyl acetate, and aqueous fractions [HF, EAF, and AQF, respectively] using ultraviolet B (UVB)-induced HaCaT cells. Materials and Methods: Solvents with different polarity were used to fractionate crude ethanolic extract of G. lemaneiformis. The physicochemical properties of HF, EAF, and AQF were detected by gas chromatography–mass spectrometry, Fourier transform-infrared spectroscopy, and high-performance liquid chromatography–mass spectroscopy. The antioxidant and antiapoptotic effects of the fractions were evaluated by estimating the levels of reactive oxygen species, antioxidant enzymes, and mitochondrial membrane potential (MMP) in UVB-exposed HaCaT cells. Results: According to our results, fatty acids, chlorophyll-a, and soluble polysaccharides were, respectively, present in the HF, EAF, and AQF. Furthermore, both EAF and AQF decreased the UVB-induced apoptosis by decreasing MMP. These results also suggest that EAF and AQF provide cytoprotective effects by activating the antioxidant enzymes. The soluble polysaccharides of AQF and chlorophyll-a of EAF were positively correlated with antioxidant activity. In addition, AQF exhibited stronger antioxidant and anti-photodamage properties than that of other fractions in UVB-radiated HaCaT cells. Conclusion: The results of this study indicated that water-soluble polysaccharides from G. lemaneiformis may be suitable to use as a natural anti-photodamage agent.